TG02, a Novel Oral Multi-Kinase Inhibitor of Cdks, JAK2 and FLT3 with Potent Anti-Leukemic Properties

TG02, a novel oral multi-kinase inhibitor of CDKs, JAK2 and FLT3 with potent anti-leukemic properties

KC Goh1,4, V Novotny-Diermayr1,4, S Hart1, LC Ong1, YK Loh1, A Cheong1,YCTan1,CHu1, R Jayaraman1, AD William1, ET Sun1, BW Dymock1, KH Ong2, K Ethirajulu1, F Burrows3 and JM Wood1

1S*BIO Pte Ltd, Singapore, Republic of Singapore; 2Tan Tock Seng Hospital, Singapore, Republic of Singapore and 3Tragara Pharmaceuticals, San Diego, CA, USA

TG02 is a novel pyrimidine-based multi-kinase inhibitor that those of the breast, colon, liver and brain.5 It is believed that inhibits CDKs 1, 2, 7 and 9 together with JAK2 and FLT3. It pharmacological inhibition of some of these kinases may dose-dependently inhibits signaling pathways downstream of provide new therapeutic options for cancer patients. In CDKs, JAK2 and FLT3 in cancer cells with the main targets being CDKs. TG02 is anti-proliferative in a broad range of tumor particular, the simultaneous inhibition of CDK1, 2 and 9 has cell lines, inducing G1 cell cycle arrest and apoptosis. Primary recently been shown to increase apoptotic killing of lung cancer cultures of progenitor cells derived from acute myeloid and osteosarcoma cells compared with the inhibition of a single leukemia (AML) and polycythemia vera patients are very CDK alone.6 sensitive to TG02. Comparison with reference inhibitors that The Janus Kinase (JAK) family consists of four protein tyrosine block only one of the main targets of TG02 demonstrate the kinases that have important roles in the cellular signaling benefit of combined CDK and JAK2/FLT3 inhibition in cell lines pathways, controlling proliferation, differentiation and apopto- as well as primary cells. In vivo, TG02 exhibits favorable 7 pharmacokinetics after oral dosing in xenograft models and sis. Among them, JAK2 has a prominent role in mediating many accumulates in tumor tissues, inducing an effective blockade of aspects of hematopoiesis from stem cells to the myriad of both CDK and STAT signaling. TG02 induces tumor regression specialized blood cells. Consequently, genetic aberrations after oral dosing on both daily and intermittent schedules in a involving JAK2 have been found to be associated with a wide murine model of mutant-FLT3 leukemia (MV4-11) and prolongs spectrum of hematological malignancies, including myelopro- survival in a disseminated AML model with wild-type FLT3 and 8 JAK2 (HL-60). These data demonstrate that TG02 is active in liferative neoplasms, lymphomas and some rare leukemias. various models of leukemia and provide a rationale for the FLT3 (fms-like tyrosine kinase-3) belongs to a family of Class III ongoing clinical evaluation of TG02 in patients with advanced receptor tyrosine kinases and is the most frequently mutated leukemias. gene in acute myeloid leukemia (AML) associated with poor Leukemia (2012) 26, 236–243; doi:10.1038/leu.2011.218; prognosis in patients.9 As myeloproliferative neoplasms and published online 23 August 2011 AML continue to present as unmet medical needs in hematol- Keywords: TG02; CDK; JAK2; FLT3; multi-kinase inhibitor; AML ogy, JAK2 and FLT3 offer hope as novel targets for the development of innovative therapies. Macrocyclic natural products possess conformationally de- fined ring structures that have evolved to interact with proteins Introduction and influence their function.10 Synthetic macrocyclic organic compounds offer great potential for pharmacological applica- Kinase inhibitors have found increasing use in clinical oncology tions, but synthetic challenges and poor pharmacologic proper- as molecularly targeted therapeutics, due largely to advances in ties have limited their application. Recent advances in synthetic our understanding of the pivotal signaling pathways that trigger medicinal chemistry are beginning to change this scenario.10 and sustain the malignant state.1 In many cases, broad-spectrum In this study, we report the pharmacological profile of TG02, a inhibitors have yielded superior efficacy compared with novel, low molecular-weight synthetic macrocycle with potent traditional agents, as exemplified by multi-kinase inhibitors inhibitory activities against several CDKs, JAK2 and FLT3. TG02 such as sunitinib2 and sorafenib.3 The utility of these drugs is exhibits favorable physico-chemical and pharmaceutical prop- often a consequence of the simultaneous targeting of multiple erties and demonstrates efficacy in in vitro and in vivo models of pathways critical for tumor growth, including those contributing hematological malignancies as well as primary cells from to proliferation, angiogenesis and apoptotic regulation. patients with erythroid and myeloid malignancies. Findings Among the best characterized pathways in relation to cellular from these studies provide the rationale for the ongoing clinical proliferation are those under the control of cyclin-dependent evaluation of TG02 in patients with advanced hematological kinases (CDKs). CDKs are serine-threonine kinases that have malignancies. important roles in cell cycle control (CDK1, 2, 4 and 6), initiation of transcription (CDK7 and 9), and neuronal develop- 4 ment (CDK5). Aberrations in the cell cycle CDKs and their Materials and methods cyclin partners have been observed in various tumors, including Compounds and reagents Correspondence: Dr KC Goh, S*BIO Pte Ltd, 1 Science Park Road, TG02, originally designated SB1317, was discovered by S*BIO #05-09 The Capricorn, Singapore 117528, Republic of Singapore. (Singapore) and licensed to Tragara Pharmaceuticals (San E-mail: [email protected] 4These authors contributed equally to this paper. Diego, CA, USA) in 2008. TG02 and TG101348 were Received 21 April 2011; revised 15 June 2011; accepted 4 July 2011; synthesized by Sai Advantium Pharma (Hyderabad, India). published online 23 August 2011 SNS-032 was synthesized by S*BIO. Sunitinib and seliciclib TG02, a multi-kinase anti-leukemic agent KC Goh et al 237 Table 1 In vitro Kinase Spectrum of TG02 Beckman Coulter Z1Particle Counter (Brea, CA, USA) and aliquoted as follows: B1 Â 105 cells for FLT3 genotyping, B 5 B 6 Kinase IC50 (nM) Source 5 Â 10 cells for FACS analysis and 3 Â 10 cells for proliferation assay. CDK9 3 Invitrogen CDK5 4 Invitrogen CDK2 5 Invitrogen CDK3 8 Millipore Animal models CDK1 9 Invitrogen Female BALB/c mice and female athymic BALB/c nude mice Lck 11 Millipore (BALB/cOlaHsd-Foxn1nu) were obtained from the Biological TYK2 14 Invitrogen Resource Centre (BRC, Biopolis, Singapore) and female NOD/ Fyn 15 Millipore SCID mice (NOD.CB17-Prkdcscid/ARC). from the Animal JAK2 19 Invitrogen Resource Centre (ARC, Murdoch, WA, Australia) and were FLT3 19 Invitrogen FLT3D835Y 21 Millipore 9–16 weeks of age at the time of tumor implantation. Standard Fms 27 Millipore protocols were followed, in compliance with the National TYRO3 36 Invitrogen Institutes of Health and National Advisory Committee for CDK7 37 Millipore Laboratory Animal Research guidelines (IACUC approval ERK5 43 CEREP #0800371). Female BALB/c nude mice were implanted sub- p38d 56 Invitrogen cutaneously in the right flank with 1 Â 107 MV4-11 cells. Cells JAK1 59 Invitrogen were resuspended in 50 ml serum-free growth medium, mixed Other kinases tested (Abl, ALK, ASK1, Aurora A, Aurora B, CaMKVI, 1:1 with Matrigel (Cat #354248; BD Biosciences, San Diego, CDK4, CDK6, Chk1, CK1a1, CK1e, CK1d, c-Raf, DYRK1B, EGFR, CA, USA) and injected in a total volume of 100 ml. Tumor FAK, GSK3a, IGF-1R, IKK-b, InsR, IRAK4, JAK3, JNK1, KDR, Kit, MAPKAP-K2, MEK1, MKK6, Nek2, p38a, PAK2, PAK3, PDGFRb, growth was monitored twice weekly. For the efficacy study, PDK1, Pim-3, PKA, PKBa, PKCe, PLK1, PLK3, PRAK, Src, TAK1, animals were randomised on day 11 after inoculation (day 0) into 3 TLK2, TTK and ZAP-70) gave less than 50% inhibition at 100 nM. treatment groups with mean tumor volumes of 146–150 mm . Values were determined from a 10-point titration in duplicates, done Mice were treated daily for 21 days with 10–40 mg/kg TG02 once. hydrochloride or vehicle (MC/Tween). For the second experiment, animals were treated with vehicle or 30 mg/kg TG02 citrate daily or 60 mg/kg TG02 citrate every fourth day. For the were obtained from Sequoia Research Products (Pangbourne, pharmacokinetic–pharmacodynamic study the average starting 3 3 UK) and LC Laboratories (Woburn, MA, USA), respectively. tumor volume was 328 mm (range 144–550 mm ). For the HL- 60 intravenous model, 1 Â 107 HL-60 cells were injected in 0.2 ml serum-free medium into the tail vein of NOD/SCID mice. In vitro kinase assays 15 days after cell inoculation, mice were randomized into 4 These assays were performed at various contract research groups and drug treatment was initiated. Treatment was organizations indicated in Table 1. Detailed protocols are administered for 5 days followed by a 5-day break (5d on 5d available from CROs on request. ATP was used at or close to Km off), for 3 cycles, using either 40 mg/kg per oral or 20 mg/kg values. intraperitoneal route. Mice were monitored for hind-leg paralysis and body weight changes daily. Mice that reached the end point (paralysis in all limbs, moribund state) were terminated. The Cell lines Prism 5 Logrank test was used to determine the statistical The transformed murine pre-B cell lines Ba/F3-JAK2V617F and significance of any difference in the survival curves between a Ba/F3-JAK2WT were generous gifts of Dr Martin Sattler treatment group and the corresponding vehicle group. All doses (Dana-Farber Cancer Institute, Boston, MA, USA). All cell lines for in vivo experiments were given as free base equivalents. were obtained from the American Type Culture Collection (Manassas, VA, USA) with the exception of SET-2, Karpas 1106P and 32D, which were purchased from DSMZ (Braunschweig, Additional methods and reagents Germany). NF114, a normal human dermal fibroblast, was The following methods and reagents are described in obtained from CellResearchCorp., Singapore. Cell lines were Supplementary Information: preparation of genomic DNA, cultured according to vendor’s instructions using One Shot fetal genotyping for FLT3-ITD, detection of JAK2V617F mutation, bovine serum from Invitrogen (Carlsbad, CA, USA). pFLT3 ELISA, flow cytometry, pharmacokinetic analysis, cell proliferation assay, primary erythroid progenitor cells, western blot assays. Primary AML blast cells Primary cellsFeither peripheral blood mononuclear cells or bone marrow mononuclear cells from AML patientsFwere Results obtained from AllCells (Emeryville, CA, USA) and ProteoGenex (Culver City, CA, USA). Cells were thawed in the AML blast TG02 demonstrates a novel kinase inhibitory spectrum expansion medium (StemSpan Serum-Free Expansion Medium TG02, a novel pyrimidine-based macrocycle (Figure 1), was from StemCell Technologies, Vancouver, BC, Canada) contain- originally discovered as a potent inhibitor of CDK2, JAK2 and ing 100 ng/ml of FLT3 ligand, 100 ng/ml of stem cell factor, FLT3. Subsequently, it was tested against a panel of 63 kinases 20 ng/ml of interleukin-3 and 20 ng/ml of interleukin-6 and in in-vitro assays (Table 1), covering all major sub-families of the plated at 2.5 Â 105 cells/ml. The cytokines were diluted 100-fold human kinome. TG02 most potently inhibited CDK isoforms, from the cytokine cocktail CC100 (StemCell Technologies). with IC50 of 3-9 nM for CDKs 1,2,3,5 and 9 and IC50 of 37 nM for Cells were diluted every 2–4 days to a density of 3 Â 105 per ml. CDK7. CDK6 was inhibited somewhat less potently, with an Between day 10 and 13, the expanded blasts were counted on IC50 of 113 nM, and CDK4 was not significantly inhibited. TG02

Leukemia TG02, a multi-kinase anti-leukemic agent KC Goh et al 238 also potently inhibited the Janus Kinase members JAK1, JAK2 sensitivity between JAK2/FLT3 wild type (first 4 lines) versus and TYK2 with IC50 between 14 and 59 nM, while sparing JAK3 mutant or activated-WT bearing cell lines (last 8 lines) with an IC50 of 4,570 nM. There was also strong activity on Class (P ¼ 0.7591). TG02 was almost always more potent than SNS- III receptor tyrosine kinases with Fms and Fms-like tyrosine 032 in all cell lines (lower IC50), but the mean fold difference in D835Y kinase 3 (FLT3) as well as its drug-resistant mutant (FLT3 ) IC50 values was significantly larger for the JAK2-dependent being inhibited with IC50 between 19–27 nM. The src family panel (4.5) compared with the non-JAK2-dependent panel (1.5- kinases Lck and Fyn were inhibited potently (11–15 nM), fold for the first 6 lines in the liquid tumor panel, 1.7 for the solid but Src itself was not inhibited at 100 nM. Additionally, TG02 tumor panel). This suggests that the additional JAK2 activity blocked the MAPK family members ERK5 and p38d with IC50 of leads to enhanced cytotoxic activity of TG02 relative to SNS- 43 and 56 nM, respectively. Taken together, these data show 032 in these JAK2-dependent lines. In the context of the JAK2- that TG02 is a multi-kinase inhibitor with a novel spectrum of dependent panel, TG02 was also significantly more potent activities. (0.14 mM) than the selective JAK2 inhibitor TG101348 (0.93 mM) (P ¼ 0.0028). TG02 inhibited the normal fibroblast in the same concentration range as the solid tumor cell lines, where potencies TG02 has potent anti-proliferative effects in tumor cell were lower than on the liquid tumor panel. lines as well as primary cells and induces cell cycle Next, the anti-proliferative activities of TG02 and reference arrest and apoptosis compounds were tested in primary diseased cells. In ex vivo To assess the effects of combining multiple targets in one expanded erythroid progenitors from polycythemia vera patients molecule, the anti-proliferative activity of TG02 was compared (n ¼ 14), TG02 significantly compromised the viability of these with SNS-032 (a CDK inhibitor without JAK2 activity11) and cells (0.047 mM) more potently than either SNS-032 (0.12 mM)or TG101348 (a JAK2 inhibitor without CDK activity12) in a 19-cell TG101348 (0.20 mM) (Figure 2b, Supplementary Table 2A). All panel consisting of 12 liquid tumor lines, 6 solid tumor lines and three drugs exhibited equivalent cytotoxic effects on erythroid 1 normal fibroblast (Figure 2a, Supplementary Table 1). In the progenitors expanded from healthy volunteers (Supplementary combined 18-tumor panel, the mean IC50 for TG02 (0.19 mM) Table 2B). was significantly lower than that for SNS-032 (0.44 mM) In expanded blast cells from AML patients (n ¼ 16, Figure 2c, (P ¼ 0.0094). TG02 showed a stronger inhibition of the liquid Supplementary Table 3), TG02 was compared with 2 CDK 11 tumor panel (0.13 mM) compared with the solid tumor panel inhibitors (SNS-032, seliciclib ) and a FLT3 inhibitor (suniti- 13 (0.30 mM), whereas SNS-032 had similar effects on both. Within nib ). Five of the expanded blast cell samples were randomly the liquid tumor panel, there was no significant difference in selected for FACS analysis, and 490% were found to express the IL-3 receptor alpha chain CD123, a marker for human AML stem cells,14 demonstrating that they were the intended population (data not shown). The mean IC50 obtained was 60 nM for TG02, 184 nM for SNS-032 and 45.6 mM for seliciclib. The number of FLT3-ITD samples was too small to make a TG02 statistically significant comparison between WT and mutated C H N O patients. Sunitinib, a FLT3 inhibitor without CDK activity, was 23 24 4 m MW = 372.46 Da less potent than TG02, with a mean IC50 of 4 M. The anti- proliferative action of TG02 was consistent with the observation of G1 cell cycle arrest in treated MV4-11 cells (Figure 3a, left panel). The fraction of apoptotic cells increased in a time- dependent manner over 72 h (Figure 3a, right panel). These data Figure 1 Structure of TG02. demonstrated that the multi-kinase activity of TG02 is associated

2.0 0.4 100

1.5 0.3 10 M) ( 1.0 0.2 1 50 IC 0.5 0.1 0.1

0.0 0.0 0.01 02 TG02 TG TG02 SNS-032 SNS-032 SNS-032 TG101348 TG101348 SeliciclibSunitinib

Figure 2 TG02 has a broad anti-proliferative effect in cell lines and primary cells. (a) Cell proliferation IC50 for various tumor cell lines (see Supplementary Table 1) was determined using the CellTiter 96 Aqueous One Solution MTS assay. Cells were seeded in log-growth phase, treated with serial dilutions of the drug in triplicates, the assay reagents were added after 48 h incubation and the IC50 values determined using XLﬁt software. The assay was repeated at least three times for each cell line. Values shown are mean IC50. The JAK2 inhibitor TG101348 was only tested against the 6 cell lines where JAK2 is implicated in proliferation. (b) Erythroid progenitors were expanded from peripheral blood mononuclear cells of 14 polycythemia vera patients (see Supplementary Table 2). The cells were grown independently in two separate experiments (triplicates for each dose). IC50 for TG02 are compared with those for the CDK inhibitors SNS-032 and the JAK2 inhibitor TG101348. (c) AML blasts were expanded from either peripheral blood mononuclear cells or bone marrow of 16 AML patients (see Supplementary Table 3). The cells were grown independently in two separate experiments (triplicates for each dose). IC50 for TG02 are compared with those for the CDK inhibitors SNS-032 and seliciclib, and the FLT3, VEGFR and pan-kinase inhibitor sunitinib.

Leukemia TG02, a multi-kinase anti-leukemic agent KC Goh et al 239 with more consistent anti-proliferative and pro-apoptotic TG02 potently inhibits CDK activity in cells activity than other compounds inhibiting subsets of these targets Studies were performed to investigate if biochemical kinase (CDKs, JAK2, FLT3). inhibition translates into pathway modulation in the cellular context. MV4-11 cells were treated for 24 h with TG02 or equipotent concentrations (in terms of proliferation) of seliciclib. G1 Arrest induced by TG02 Apoptosis induced by TG02 The mean IC50 for pRb inhibition was 125 nM for TG02, 25 100 compared with 19.8 mM for seliciclib (Figure 4a, Supplementary Table 4). At the higher concentrations of both compounds, cells 20 80 were undergoing apoptosis, resulting in less detectable actin 15 60 (beyond 0.8 and 80 mM, respectively). The IC50 of TG02 against 10 40 pFLT3 in cells (3 h treatment) was 4,700 nM and 59 nM for sunitinib (Figure 4b, Supplementary Table 4). In the FLT3-ITD- 5 20 dependent cell lines such as MV4-11, STAT5 has been described % of apoptotic cells Ratio of G1/G2 cells 0 0 to be directly downstream of the FLT3-ITD mutant and 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 15 independent of JAK activity. The mean IC50 of TG02 for pSTAT5 was 560 nM and 50 nM for sunitinib. It is not known why the IC50 values for pFLT3 versus pSTAT5 inhibition are consistent for sunitinib, but 48-fold different for TG02. A possible explanation is that TG02 is inhibiting other kinase(s) that phosphorylate STAT5. In summary, TG02 appeared to directly inhibit CDK2 more potently than FLT3 in MV4-11 cells. The effects on JAK-STAT signaling were studied in Karpas 1106P (hyper-activated wild-type JAK2)16 and SET-2 cells (constitutively phosphorylated JAK2V617F)17 and compared with 24 h DMSO 24 h 0.1 M TG02 TG101348. In Karpas 1106P, the IC50 for pJAK2 (Y1007/8) and Figure 3 TG02 induces cell cycle arrest and apoptosis in MV4-11 pSTAT3 inhibition by TG02 was 63 and 53 nM, respectively, cells. (a) MV4-11 cells were treated with 100 nM TG02 for the times compared with 150 and 90 nM for TG101348 (Figure 4c, indicated. Cells were ﬁxed with ice-cold ethanol, and the DNA Supplementary Table 4). Phospho-JAK2 (Y1007/8) levels were content was determined by propidium iodide staining. Error bars increased by both drugs in SET-2 cells, consistent with previous indicate standard deviations from three individual experiments. V617F (b) MV4-11 cells were treated with 100 nM TG02 for 24 h, stained reports of similar observations in JAK2 -expressing cell 18,19 with the Annexin V-FITC apoptosis detection kit and analyzed on a lines. The IC50 for STAT3 inhibition in SET-2 cells was FACS Calibur. Examples of dot blots obtained are shown. 250 nM for TG02 and 110 nM for TG101348 (Figure 4d,

TG02 Sel. Seliciclib TG02 TG101348 M M M M M M M M M M M M M M M M μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ 0.05 µM 0.10 µM DMSO 0.1 0.2 0.4 0.8 1.0 control DMSO 0.15 µM 0.25 µM 0.50 µM 20 DMSO 0.2 0.4 10 20 40 80 100 DMSO 0.1 0.8 1.0 pRb pJAK2

Rb pSTAT3

β-actin β-actin

MV4-11 Karpas 1106P

TG02 Su. Sunitinib TG02 TG101348 M M M M M M M M M M M M μ μ μ μ μ μ M M M M M μ μ μ μ μ M M M M μ μ μ μ μ μ μ μ μ μ DMSO 1 2 4 5 10 0.8 DMSO 0.1 0.2 0.4 0.8 1.0 0.15 0.8 0.25 0.50 DMSO 0.1 0.2 0.4 1.0 control* DMSO 0.05 0.10 pFLT3 pJAK2

pSTAT5 pSTAT3

β-actin β-actin

MV4-11 SET-2

Figure 4 Effects of TG02 on the target signaling pathways. Western blots were performed with antibodies indicated. Each western blot was done at least twice using cell lysates prepared at different times from different passages of cells (between passage 2–15). IC50 were determined using densitometric analysis. Representative western blots are shown for each experiment. (a) MV4-11 cells were treated with drugs for 24 h and probed for pRb and Rb. (b) MV4-11 cells were treated with drugs for 3 h and probed for pFLT3 and pSTAT5. (c) Karpas 1106P cells were treated with drugs for 3 h and probed for pJAK2 and pSTAT3. (d) SET-2 cells were treated with drugs for 3 h and probed for pJAK2 and pSTAT3. All blots were re-probed for b-actin for loading comparison; Sel, seliciclib; Su, sunitinib.

Leukemia TG02, a multi-kinase anti-leukemic agent KC Goh et al 240

TG02 (μM) SNS-032 (μM) TG02, 0.3 μM 2 h 4 h 6 h DMSO 0.03 0.3 3 DMSO 0.03 0.3 3

pRNA pol II S2 DMSO TG02 DMSO TG02 DMSO TG02

cdc6 pRNA pol II S2

cyclin E Mcl-1

Mcl-1 cleaved PARP

Bcl2 α-tubulin cleaved PARP HL-60 β-actin

HL-60

Figure 5 Effects of TG02 on cell cycle and apoptotic markers. Western blots were performed with antibodies indicated. Each western blot was done at least twice using cell lysates prepared at different times from different passages of cells. Representative western blots are shown for each experiment. (a) HL-60 cells were treated for 4 h with drugs and probed for RNA Pol II Ser 2, cdc6, cyclin E, Mcl-1, Bcl2 and cleaved PARP. (b) Time course for the effects of TG02 on RNA Pol II Ser 2, Mcl-1 and PARP cleavage in HL-60 cells.

Supplementary Table 4). Overall these data show that TG02 60 mg/kg dosing, the mean plasma concentration of TG02 B effectively blocks intracellular phosphorylation of substrates for remained above cellular IC50 in MV4-11 for 8 h. Drug levels CDK2, JAK2 and FLT3 at sub-micromolar concentrations. in the tumors remained above the IC50 for MV4-11 cells for 8 and 24 h for the low and high dose groups, respectively. Figures 6a and b showed that TG02 was selectively retained at TG02 induces markers of G1 arrest and apoptosis supra-therapeutic levels in tumor tissues. To further analyze functional changes in cells, HL-60 cells were To assess target engagement by TG02 in tumor tissue, MV4-11 treated with TG02 for 3 h. TG02 blocked mRNA transcription as tumor lysates were analyzed after tumor-bearing mice were demonstrated by a dose-dependent decrease in the phosphor- treated with a single dose of 60 mg/kg. Levels of phospho-RNA ylation of RNA polymerase II Ser2 (Figure 5a, top panel). poly II Ser2 were used as a read-out for CDK9 inhibition. Whereas SNS-032 inhibited phosphorylation of RNA pol II Ser2 Inhibition was observed from 0.5 h, reached a maximum (100%) at 300 nM, TG02 had a marked effect at concentrations as low as at 6 h and showed total recovery by 24 h (Figure 6c, top panel). 30 nM, indicating that CDK9 is the most sensitive target of the The pRb signal, read-out for CDK2, was inhibited by B30% drug as reﬂected by Table 1. TG02 also led to dose-dependent after 0.5 h, reduced by 490% between 6–24 h and returned to decrease in cdc6 levels (Figure 5a, 2nd panel from top), a pre-treatment levels after 48 h (Figure 6c, second from top). 20 protein that is stabilized through phosphorylation by CDK2. Levels of pSTAT5 (Y694) were assessed as a read-out for FLT3 Cyclin E, a cyclin partner of CDK2 that is degraded on S-phase inhibition. Maximum inhibition was observed at 3–6 h, but the entry, showed increased levels after treatment with 0.3–3 mM signal recovered beyond vehicle baseline at 16–24 h. In a TG02 (Figure 5a, 3rd panel from top), supporting the G1 arrest separate experiment, the effects of a single acute dose of TG02 shown in Figure 3a. Levels of the anti-apoptotic Mcl-1 were citrate (30, 40 or 60 mg/kg) on pFLT3 (Y591) levels in the tumor downregulated whereas levels of the pro-apoptotic Bcl-2 lysates was assessed by ELISA. All three dose levels showed remained unchanged in TG02-treated samples. Cell death was effective reduction in pFLT3, with maximum reductions (12 h induced by TG02 at concentrations X300 nM as indicated by an post-dose) of 23.5, 51.5 and 65% from baseline levels shown in increase in cleaved poly (ADP ribose) polymerase (PARP, animals treated with 30, 40 and 60 mg/kg, respectively Figure 5a, 2nd panel from the bottom). Changes in RNA pol II (Figure 6d). Apoptosis, as exempliﬁed by the increased levels Ser2, Mcl-1 and cleaved PARP were time-dependent in HL-60 of cleaved PARP (Figure 6c second from the bottom), occurred cells (Figure 5b). In summary, these data show that TG02 between 6 and 24 h. Taken together, these data show that TG02 exposure leads to CDK9 inhibition, followed by G1 phase arrest effectively inhibits CDK2, CDK9 and FLT3 in vivo, causing and apoptotic induction. apoptosis in the tumor tissues.

TG02 is enriched in tumor tissues and shows effective target engagement TG02 is efﬁcacious in animal models of AML The pharmacokinetics (PK) of TG02 was determined in MV4-11 The efﬁcacy of TG02 was initially tested in a subcutaneous (s.c.) tumor-bearing nude mice after a single oral dose of 30 or AML model (MV4-11). The average tumor volume at start was 60 mg/kg (Figures 6a and b, Supplementary Table 5). Maximal 87 mm3. Mice were dosed orally daily for 21 days with 10, 20 or plasma concentrations (Cmax) were 74 and 154 ng/ml for 30 and 40 mg/kg TG02, leading to a tumor growth inhibition of 53%, 60 mg/kg, respectively, measured at 0.5 h post-dose (Tmax). At 61% and 113%, respectively (Figure 7a). Treatment was well

Leukemia TG02, a multi-kinase anti-leukemic agent KC Goh et al 241

10,000 30 mg/kg TG02 10,000 60 mg/kg TG02

1,000 1,000

100 IC50 (MV4-11)

100 IC50 (MV4-11) 10 or ng/g ( ± S.E.M.)

10 or ng/g (± S.E.M.) 1 Mean concentration, ng/mL

Plasma (ng/mL) Mean concentration, ng/mL Plasma (ng/mL) 1 Tumor (ng/g) 0.1 Tumor (ng/g) 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Time (h) Time (h)

60 mg/kg TG02 0.45 30mg/kg 0.40 40mg/kg veh. 0.5 h 3h 6 h 16 h 24 h 48 h 72 h 0.35 60mg/kg pRNA pol II S2 0.30 0.25 pRb (S807/811) 0.20

pSTAT5 (Y694) 0.15 0.10 pFLT3 (Abs. 450 nM) cleaved PARP 0.05 0.00 β-actin 0 12 24 36 48 60 72 Time (h)

Figure 6 Pharmacokinetic and pharmacodynamic analysis of TG02 in plasma and tumor tissues from MV4-11 tumor-bearing mice. Concentrations of TG02 in the plasma or tumor tissue of MV4-11 tumor-bearing nude mice, after single oral doses of (a) 30 mg/kg and (b)60mg TG02 (citrate salt) were determined using high performance liquid chromatography mass spectrometry analysis. The dotted line shows the IC50 of TG02 on MV4-11 proliferation. Data is mean with error bars showing standard error of the means. (c) Pharmacodynamic response in tumor tissue after a single dose of 60 mg/kg TG02. Representative western blots from 25 mg tumor lysates are shown. (d) Pharmacodynamic response after a single dose of 30, 40 or 60 mg/kg TG02. Equal amounts of total protein lysates from MV4-11 tumors were assayed in duplicates using an ELISA against pFLT3 (Y591) and compared with tumor lysates from untreated mice (average absorbance of 0.335 at 0 h). Data shown are averages±s.d. from six mice.

TG02 HCl salt TG02 HCl salt 100 600 Vehicle TG02 10 mg/kg q.d. 500 TG02 20 mg/kg q.d 80 TG02 40 mg/kg q.d 400 TGI = 53% 60 300 ) ± S.E.M.

3 * 40 200 Percent survival (mm TGI = 61% 20

Mean Tumor Volume 100 TGI = 113% 0 0 ***

0 3 6 9 12 15 18 21 30 40 50 60 70 80 Days Days

TG02 citrate salt Vehicle Group 800 TG02 30 mg/kg q.d Vehicle 5d on 5d off (p.o.) TG02 60 mg/kg Q4Dx6 TG02 40 mg/kg 5d on 5d off (p.o.) 600 Vehicle 2d on 5d off (p.o) TG02 100 mg/kg 2d on 5d off (p.o.) 400 TGI = 86% ) ± S.E.M.

3 Vehicle 5d on 5d off (i.p.) ** TG02 20 mg/kg 5d on 5d off (i.p.)

(mm 200 ** TGI = 86% Mean Tumor Volume 0

0 3 6 9 12 15 18 21 Days

Figure 7 TG02 is efficacious in in vivo models of AML. (a) MV4-11 tumor-bearing nude mice were randomized into four groups of ten animals each. Mice were treated daily for 21 days with TG02 hydrochloride salt or vehicle (MC/Tween). Tumor growth inhibition (TGI) is indicated on the right hand side. The maximum BW loss for the 100 mg/kg group was 11.3%, whereas no significant BW loss was found in any of the other groups. ANOVA with Dunnett’s post test was performed. *Denotes Po0.05, **denotes Po0.01, ***denotes Po0.001. (b) NOD/SCID mice (9 animals per group, except for the per oral 5d on 5d off vehicle group with 10 animals) were injected with HL-60 cells intravenous Treatment was started on day 15 and lasted for 30 days. Per cent survival was shown on this Kaplan-Meier plot. The Logrank test (analyzing medians) was used to determine the statistical significance of any difference observed in the survival curves between a treatment group and the corresponding vehicle control group. (c) The experiment was performed as described under A, except that n ¼ 12/group and TG02 citrate salt was used.

Leukemia TG02, a multi-kinase anti-leukemic agent KC Goh et al 242 tolerated with no body weight loss at termination (Supplemen- potent against JAK2 and TYK2. Importantly, TG02 demonstrated tary Table 6). Tumors had regressed completely in 4/10, 2/10 B240-fold selectivity for JAK2 relative to JAK3. As JAK3 and 8/9 mice for the 10, 20 and 40 mg/kg groups, respectively. deficiency has been shown to lead to severe immune disorders In a second AML model (HL-60 intravenous), vehicle-treated in humans,26 it is desirable to avoid inhibiting this JAK isoform. NOD/SCID mice developed bilateral hind leg paralysis, a The interesting CDK-JAK2-FLT3 inhibitory profile of TG02 hallmark symptom of this disease model, between days 29 suggests therapeutic potential for treating a broad range of and 41, which was accompanied by progressive body weight hematological malignancies. The other kinase targets of TG02 loss due to disease. The median survival of the animals of the 3 (Table 1), including ERK5, TYRO3 and p38d, may further vehicle groups was between 39–42 days. All mice in the vehicle broaden therapeutic indications to a wide range of solid tumors, groups reached the endpoint (as described in Materials and if they are confirmed in a cellular and functional context. methods) at the latest by day 53. Mice treated, using a 40 mg/kg TG02 demonstrated effective inhibition of CDK and JAK2 per oral 5d on 5d off Â 3-cycle regimen, had a median survival signaling pathways in the relevant cell models examined. It was of 48 days, which translated to a 9-day protection compared much more potent than seliciclib in inhibiting phosphorylation with the corresponding vehicle group (p o0.001). Mice treated of Rb protein. In wild-type JAK2-bearing Karpas 1106P cells, with 100 mg/kg per oral 2d on 5d off Â 4-cycle regimen had a both pJAK2 and pSTAT3 were strongly inhibited. In the median survival of 59 days, increasing their median survival by JAK2V617F-bearing cell line SET2, pSTAT3 was dose-depen- 19 days compared with the corresponding vehicle group dently inhibited, although pJAK2(Y1007/1008) levels were (Po0.001). Mice treated with a 20 mg/kg intraperitoneal 5d on increased. Based on molecular modeling, we speculate that 5d off Â 3-cycle regimen showed the best efficacy with a median TG02 preferentially binds to and stabilizes a ‘pre-activated’ form survival of 68 days, amounting to a 26-day protection of JAK2V617F, already phosphorylated at Y1007/8, leading to an (Po0.0001) (Figure 7b). Notably, 4/9 mice in this group did accumulation of this phosphorylated JAK2V617F species. Such a not develop hind-leg paralysis at all during the study. Taken scenario is similar to what others have proposed after together, these data demonstrated the efficacy of TG02 in two independent observations with structurally unrelated JAK2 different models of AML. inhibitors in other JAK2V617F-bearing cell lines.18,19 In MV4-11 As the citrate salt of TG02 was chosen as the preferred form for cells, TG02 showed inhibition of pFLT3 and pSTAT5, but at a further development studies, the MV4-11 s.c. efficacy model was higher concentration than sunitinib, consistent with the in vitro repeated with citrate salt to investigate new dosing regimens. In potencies of these 2 compounds. However, the much higher this second study, treatment was initiated when tumors were IC50 for pFLT3 inhibition (4.7 mM) compared with the cytotoxic 3 larger than in the first study (average size of 147 mm ). Mice were dose (0.16 mM) poses the question of the extent to which FLT3 treated with either 30 mg/kg daily or 60 mg/kg TG02 every fourth impacts proliferation relative to CDK. day (Q4D) Â 6 days, starting on day 0. The tumor growth In HL-60 cells, the blockade in signaling pathways translated inhibition achieved on day 21 was 86% for both schedules into downstream processes that reflected transcriptional inhibi- (Po0.01) (Figure 7c). The average tumor weight in the treated tion (decrease in phosphorylation of RNA pol II Ser2), failure of groups was significantly (Po0.001) reduced by 75 and 84% S-phase entry (cyclin E accumulation) and apoptotic induction compared with vehicle-treated animals for the 30 and 60 mg/kg (depletion of Mcl-1, and cleavage of PARP). Inhibition of the groups, respectively (data not shown). Both schedules were well oncogenic signaling pathways by TG02 resulted in functional tolerated with no BW loss at the end of study (Supplementary consequences for the treated cells. Dose- and time-dependent Table 6). The two dosing schedules were not significantly apoptotic induction, probably due to depletion of the short-lived different in their efficacy on either tumor volume or weight. anti-apoptotic protein Mcl-1 as a result of the transient transcriptional block caused by inhibition of CDK9, and cell cycle arrest were observed in MV4-11 and HL-60 cells. Taken Discussion together, these data demonstrated that TG02 exerted potent inhibition on its main target kinases to induce cell cycle arrest Advanced hematologic malignancies, including AML, constitute and cell death. an area with unmet medical need, and recent advances in the Importantly, comparison of TG02 with reference inhibitors understanding of their molecular etiologies engendered hopes of that block only one of the kinase targets (CDK only or JAK2 only) targeted therapies.21 Among the genetic aberrations identified in demonstrated the significant benefit of combined CDK and JAK2 hematologic malignancies are FLT3, JAK2 and the CDKs. The targeting. In both JAK2-dependent tumor cell lines and erythroid specific indications that may be suitable for these targets include progenitors from polycythemia vera patients, our data revealed (a) chronic lymphocytic leukemia, multiple myeloma and the higher efficacy of TG02 compared with SNS-032 and mantle cell lymphoma, where CDK inhibitors have shown TG101348, attesting to the advantage of co-targeting CDKs and preliminary clinical efficacy; (b) acute myeloid leukemia, where additional kinases. We attribute the equivalent cytotoxicity the FLT3-ITD mutation is present in B30% of the patients9 and against normal erythroid progenitors to the presence of activated JAK2 is an emerging target in both circulating blasts erythropoietin in the expansion medium, contributing to the and tumor stem cells;22,23 and (c) myeloproliferative diseases JAK2 dependence of expanded cells. SB1518, a JAK2 inhibitor, where JAK2 lesions were identified in 450% of the patients.24 that also showed equipotent cytotoxic effects on expanded In this study, we present the preclinical evaluation of a novel progenitors from healthy or polycythemia vera subjects, can be chemical entity, TG02, a small molecule that inhibits these administered to patients with a good therapeutic window.27 The oncogenic kinases. clinical testing of TG02 is still underway to determine whether a TG02 is a broad-spectrum CDK inhibitor, with inhibitory therapeutic window can be achieved in leukemic patients. activity against CDK1, 2, 7 and 9. This spectrum is noteworthy Additionally, TG02 was superior to both SNS-032 and the because the combined inhibition of transcriptional CDKs with FLT3 inhibitor sunitinib against primary blasts from AML cell-cycle regulatory CDKs has been shown to be advantageous patients. This approach has the effect of delivering a combina- for effective apoptotic induction in tumor cells.25 Within the tion therapy in a single molecule and is a promising drug Janus kinase family, in vitro kinase data show TG02 to be most discovery platform for the future.

Leukemia TG02, a multi-kinase anti-leukemic agent KC Goh et al 243 TG02 demonstrated favorable oral pharmacokinetic proper- treatment of a murine model of JAK2V617F-induced polycythemia ties in mouse tumor models. It was retained in tumor masses at vera. Cancer Cell 2008; 13: 311–320. supra-therapeutic levels for 8–24 h, depending on the dose. 13 Atkins M, Jones CA, Kirkpatrick P. Sunitinib maleate. Nat Rev Drug Consequently, pathway biomarkers were suppressed for sus- Discov 2006; 5: 279–280. 14 Jordan CT, Upchurch D, Szilvassy SJ, Guzman ML, Howard DS, tained periods after dosing. These translated to anti-tumor Pettigrew AL et al. The interleukin-3 receptor alpha chain is a efficacy and survival benefits in both FLT3-dependent (MV4-11) unique marker for human acute myelogenous leukemia stem cells. and independent (HL-60) models of AML, using either daily or Leukemia 2000; 14: 1777–1784. intermittent regimen. 15 Choudhary C, Brandts C, Schwable J, Tickenbrock L, Sargin B, In summary, TG02 is a novel chemical entity with broad- Ueker A et al. Activation mechanisms of STAT5 by oncogenic spectrum activity against oncogenic CDKs, with the additional Flt3-ITD. Blood 2007; 110: 370–374. 16 Melzner I, Weniger MA, Bucur AJ, Bruderlein S, Dorsch K, Hasel C benefit of targeting JAK2 and FLT3, the two kinases that are key et al. Biallelic deletion within 16p13.13 including SOCS-1 in to the etiology of several hematopoietic malignancies. Com- Karpas1106P mediastinal B-cell lymphoma line is associated with pared with mono-targeted reference inhibitors, TG02 is more delayed degradation of JAK2 protein. Int J Cancer 2006; 118: consistently anti-proliferative across tumor cell lines as well as 1941–1944. primary diseased cells. TG02’s unique kinase spectrum and 17 Uozumi K, Otsuka M, Ohno N, Moriyama T, Suzuki S, favorable pharmacological profile provide the rationale for its Shimotakahara S et al. Establishment and characterization of a new human megakaryoblastic cell line (SET-2) that spontaneously current evaluation in patients with advanced leukemias. matures to megakaryocytes and produces platelet-like particles. Leukemia 2000; 14: 142–152. 18 Grandage VL, Everington T, Linch DC, Khwaja A. Go¨6976 is a Conflict of Interest potent inhibitor of the JAK 2 and FLT3 tyrosine kinases with significant activity in primary acute myeloid leukaemia cells. Br J FB is an employee of Tragara Pharmaceuticals. All other authors, Haematol 2006; 135: 303–316. except KHO, are employees of S*BIO. 19 Tyner JW, Bumm TG, Deininger J, Wood L, Aichberger KJ, Loriaux MM et al. 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