Pan-PIM Kinase Inhibition Provides a Novel Therapy for Treating Hematologic Cancers

Published OnlineFirst January 28, 2014; DOI: 10.1158/1078-0432.CCR-13-2062

Clinical Cancer Cancer Therapy: Preclinical Research

Pablo D. Garcia1, John L. Langowski1, Yingyun Wang1, Min Chen1, Joseph Castillo1, Christie Fanton1, Marjorie Ison1, Tatiana Zavorotinskaya1, Yumin Dai1, Jing Lu1, Xiao-Hong Niu1, Stephen Basham1, Julie Chan1, Jianjun Yu1, Michael Doyle1, Paul Feucht1, Robert Warne1, Jamie Narberes1, Tiffany Tsang1, Christine Fritsch6, Audrey Kauffmann6, Estelle Pﬁster6, Peter Drueckes7, Joerg Trappe7, Christopher Wilson5, Wooseok Han2, Jiong Lan2, Gisele Nishiguchi2, Mika Lindvall2, Cornelia Bellamacina2, J. Alex Aycinena4, Richard Zang3, Jocelyn Holash1, and Matthew T. Burger2

Abstract Purpose: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies.

Experimental Design: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low Km for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context. Results: LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model. Conclusions: We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies. Clin Cancer Res; 20(7); 1–12. 2014 AACR.

Introduction estimated 70,130 cases of non-Hodgkin lymphoma (NHL), As a group, hematologic cancers are the fourth most 21,700 cases of multiple myeloma, 16,060 cases of chronic common cancer type in the United States (1). In 2012, an lymphocytic leukemia (CLL), and 13,780 cases of acute myelogenous leukemia (AML) were diagnosed (1). In spite of considerable advances with novel therapeutics, including Authors' Affiliations: 1Oncology Disease Area Research; 2Global Discov- monoclonal antibodies, stem cell transplantation, and tar- 3 4 ery Chemistry/Oncology and Exploratory Chemistry; MAP Group; Chem- geted therapies, the number of patients succumbing to these ical and Pharmaceutical Profiling Group, Novartis Institutes for Biomedical Research, Emeryville, California; 5Developmental Molecular Pathways, diseases remains high, with more than 45,000 deaths esti- Novartis Institutes for Biomedical Research, Cambridge, Massachusetts; mated in 2012 (1). 6Oncology Disease Area Research; and 7Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Basel, Switzerland In both hematologic cancers and solid tumors, therapies that target cancer drivers have been shown to be clinically Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). beneficial. With this in mind, we identified the PIM family of protooncogenes as a suitable target given that they are Corresponding Author: Pablo D. Garcia, Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608. Phone: 510-923- highly expressed in hematologic malignancies. Although 7680; Fax: 510-655-9910/510-923-2502; E-mail: gene expression alone is not always a fair predictor of cancer [email protected] relevance, PIM kinases display a few unique features. Once doi: 10.1158/1078-0432.CCR-13-2062 properly folded, PIM kinases require no further posttrans- 2014 American Association for Cancer Research. lational modifications for their activity (2); indeed, the

www.aacrjournals.org OF1

Garcia et al.

in mice. Taken together, these findings establish PIM Translational Relevance kinases as an attractive pharmacologic target in cancer Here, we describe detailed biochemical, cellular, and therapy. pharmacologic properties of a highly potent and selective inhibitor (LGB321) of the three PIM kinases. When Materials and Methods tested in more than 500 cancer cell lines of diverse Sources of gene expression data origins, we demonstrated almost exclusive antiprolifera- All gene expression microarray datasets were retrieved tive activity in cells from hematologic lineages, including from public depositories, including Gene Expression Omni- multiple myeloma, acute lymphoblastic leukemia bus (http://www.ncbi.nlm.nih.gov/geo/) and ArrayExpress (ALL), acute myelogenous leukemia (AML), and non- (http://www.ebi.ac.uk/arrayexpress/). Only human prima- Hodgkin lymphoma (NHL). This finding correlated with ry tumor or normal tissue samples that were hybridized on higher levels of PIM kinases expression in these cancers, Affymetrix human genome U133 Plus 2.0 arrays and were as compared with solid tumor cancers. To our knowl- obtained from patients that did not receive any treatment edge, LGB321 is the first Pan-PIM inhibitor with activity before tissue resection were selected. After further removing on PIM2-dependent multiple myeloma cells. LGB321 redundant or poor-quality samples, 18,960 samples across was effective in inhibiting tumor growth in an AML 40 tissue types were processed for data analysis. xenograft model, in addition to a previously reported multiple myeloma model. Our findings suggest that Microarray data processing and analysis single-agent activity for Pan-PIM inhibitors is likely to To estimate expression level of each gene, microarray raw be observed in hematologic malignancies. On the basis CEL files were processed using the Michigan custom CDF of these findings, we initiated early clinical testing of our (HGU133Plus2_Hs_ENTREZG, version 14; ref. 15) with an development candidate in multiple myeloma, empha- extended reference-based RMA summarization method sizing the translational relevance of the present work. (16). Fourteen-hundred samples from the entire data set were randomly selected to estimate parameters of the RMA fit, which were then applied directly to the remaining microarray samples to produce gene-level expressions. After crystal structure of PIM1 supports a model in which the data processing, expression levels of PIM1, PIM2, and PIM3 unphosphorylated protein adopts an active conformation were extracted for the samples described in Fig. 1 and (3). In addition to transcriptional regulation, PIM kinases Supplementary Fig. S1 and then visualized by boxplots in are phosphorylated and autophosphorylated to regulate R (http://www.R-project.org). their stability (2). Thus, the cellular activity of each PIM kinase is primarily regulated by the balance of synthesis and degradation of the protein itself (2), suggesting that expres- Biochemical assays for PIM kinases and determination sion alone may be a useful predictor of where PIM kinases of LGB321 inhibition constants are active. PIM1 (Invitrogen; PV3503), PIM2 (Invitrogen; PV3649), The PIM kinases have roles in cell-cycle progression, cell and PIM3 (Novartis) enzyme reactions were run in 50 survival, and tumorogenesis (4). PIM1 was originally dis- mmol/L Hepes, pH 7.5, 5 mmol/L MgCl2, 0.05% bovine covered as an oncogene by frequent proximal proviral serum albumin (BSA), 1 mmol/L dithiothreitol buffer with insertions of murine leukemia virus in lymphomas of a biotinylated BAD peptide (b-RSRHSSYPAGT_NH2) and infected mice (5, 6). Similar unbiased insertional mutagen- ATP substrates. Reaction products were measured using the esis screens were used to demonstrate the oncogenic poten- PerkinElmer AlphaScreen IgG Detection Kit (protein A) tial of PIM2 (7) and PIM3 (8) in PIM1 and PIM1/2 knock- with anti-phospho-(Ser/Thr) AKT substrate antibody from out mice, respectively, suggesting that PIMs may have Cell Signaling Technology. Apparent ATP Km calculations functional similarities in oncogenesis. These studies also were performed with Prism 5 (GraphPad Software Inc.) demonstrated that PIM kinases in conjunction with c-Myc using the Michaelis–Menten equation. Ki measurements result in synergistic induction of cancer (9). were performed at high ATP concentrations. For PIM1, the Here, we describe LGB321, a potent and selective ATP- ATP concentration was 2,800 mmol/L, for PIM2 it was 500 competitive small-molecule inhibitor of all three PIM mmol/L, and for PIM3 it was 2,500 mmol/L. The apparent Ki kinases (Pan-PIM kinase inhibitor). LGB321 is unique from these measurements was calculated using the Morri- relative to previously described PIM inhibitors (10–13), in son equation and converted to a true Ki using the Cheng– that it is active in PIM2-dependent cell lines (14), a kinase Prusoff relationship for an ATP site–competitive mecha- that has proven difficult to inhibit in the cellular context. nism. Active site enzyme titrations were performed on all Consistent with its activity on all three PIM kinases, LGB321 three enzyme isoforms for use in the Morrison equation. inhibits proliferation of a number of cell lines derived from Morrison equation: diverse hematologic malignancies, including multiple mye- V =V ¼ ½ðE þ I þ K Þ ð ðE þ I þ K ÞÞ loma, AML, CML, and B-cell NHL. In vivo, the compound is i o 1 i app sqrt sqr iapp ð E IÞ=ð EÞ orally available, demonstrates efficacy in tumor xenografts 4 2 : K ¼ K ð þ S=K Þ and is well tolerated within the therapeutic exposure range Cheng--Prusoffequation iapp i 1 m

OF2 Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research

Pan-PIM Kinase Inhibition in Hematologic Cancers

500 PIM1

400

300

200

100

1,000 PIM2

800

600

400 Expression intensity 200

3,500 PIM3

3,000

2,500

2,000

1,500

1,000

500

0 MM ALL AML Liver Lung Breast DLBCL marrow Prostate Stomach Pancreas Normal bone

Figure 1. mRNA expression levels in cancer tissues. PIM1, PIM2, and PIM3 expression in patient samples from the major hematologic cancers subtypes [ALL, N ¼ 350; AML, N ¼ 2,049; DLBCL-NHL, N ¼ 640; and multiple myeloma (MM), N ¼ 982] as compared with the expression in normal bone marrow (N ¼ 81). For comparison purpose, a few representative solid tumors are included: breast (N ¼ 2,422), liver (N ¼ 132), lung (N ¼ 973), pancreas (N ¼ 128), prostate (N ¼ 193), and stomach (N ¼ 101) cancers. Expression intensity was determined as described in Materials and Methods. The median expression of each gene is represented by the black center line within each box, and the ﬁrst and third quartiles are depicted by the edges of the box. The whiskers extending from each box indicate expression values that are within 1.5 times the interquartile range (IQR) from the upper or lower quartile. Outliers that are at a distance of greater than 1.5 IQR from the box are plotted individually as plus signs. www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 OF3

Garcia et al.

Biochemical kinase specificity profile The activity of LGB321 was also tested in the CCLE screen The kinase specificity profile for LGB321 was determined (18) and further testing was performed on an expanded as previously described (17). Briefly, protein kinase activity panel of hematologic cell lines. Cell lines were obtained was assayed using either the LanthaScreen (www.invitro- from commercial sources (ATCC or DSMZ) and were cul- gen.com) or the Caliper (www.caliperls.com) technologies. tured in RPMI or IMDM plus 10% to 20% FBS (Invitrogen) The biochemical activity for lipid kinases PI3Ka (phosphoi- as supplier recommended. All cell lines were thawed from nositide 3-kinase a), PI3Kb, Vps34, and PI4Kb was deter- frozen stock, grown at 37 C, 5% CO2, 95% relative humid- mined by a luminescence assay based on ATP consumption ity and cultured in T75 flasks using standard culture tech- (KinaseGlo; www.promega.com) with phosphoinositol as niques. They were expanded for at least two passages before the substrate, whereas PI3Kg, PI3Kd were determined by the being added in assay microtiter plates. Cell count was Adapta TR-FRET technology (www.invitrogen.com). For measured using a CASY Model TT counter (Roche Applied determination of the biochemical activity of recombinant Science). All cell lines were tested for and shown to be free of mTOR an antibody-dependent TR-FRET assay was used mycoplasma using PCR detection. In addition, cell line with 4EBP1 as substrate as well as an "ATP-binding assay," identity was verified by single-nucleotide polymorphism which measures the occupancy of compounds in the ATP genotyping. Cell lines were dispensed into 384-well plates site of mTOR. (Greiner Bio-One; #781098) with a final volume of 25 mL and concentrations ranging from 250 to 4,000 cells per well Cell lines and reagents in duplicate plates. Cell viability was assessed 3 hours after The KMS-11.luc human multiple myeloma tumor cell line, seeding in the first plate (start value) and 120 hours after a KMS-11 clone expressing firefly luciferase, was obtained seeding in the second plate by adding 25 mL Cell Titer-Glo from the University Health Network (UHN; Toronto, (Promega; #G7571) per well. The doubling time was cal- Ontario, Canada). KMS-11.luc and OPM-2 (DSMZ, Ger- culated for each condition and the optimal seeding density many) were cultured in RPMI-1640 (American Type Culture leading to the shortest doubling time was selected for Collection, ATCC), supplemented with 10% FBS. AML cell further profiling. Three hours after seeding, compounds lines KG-1 (ATCC) and MOLM-16 (DSMZ, Germany) were were transferred to the cells by delivering 5 mL per well of cultured in Iscove’s modified Dulbecco’s medium (IMDM; the intermediate dilution using the Velocity-Bravo Auto- ATCC) or RPMI-1640 (ATCC), respectively, supplemented mated Liquid Handling Platform (Agilent). This resulted in with 20% FBS. The AML cell line P31/FUJ (Health Sciences a final compound concentration range of 10 to 0.01 mmol/L Research Resources Bank, Japan) was cultured in RPMI-1640 in a final volume of 25 mL and a final DMSO concentration (ATCC) media supplemented with 10% FBS. All cell culture of 1%. The cell–compound mixture was incubated for 120 media for in vitro work was additionally supplemented with hours. Cell Titer-Glo was added and luminescence was read 20 mmol/L glutamine, 1,000 IU/mL penicillin and 1,000 mg/ on a Magellan plate reader (TECAN). On all plates, wells mL streptomycin. The origin and in vitro methods for the 947 containing vehicle only were included. Cell lines were independent cancer cell lines in Cancer Cell Line Encyclope- seeded in parallel into a 384-well plate (Corning; #732- dia (CCLE) has been previously reported (18). Before implan- 5528) in the same conditions and Cell Titer-Glo was added tation in vivo, KMS-11.luc and KG-1 cells were cultured in 3 hours after seeding to evaluate cell viability before treat- Dulbecco’s Modified Eagle Medium (Corning) plus 10% FBS ment (start value). Start values were subtracted and raw and 1% L-glutamine (Corning). values were percentage normalized on a plate-by-plate basis such that the median of the neutral control wells (i.e., Cellular proliferation assays DMSO) is 100% growth and the D0 wells (optical density To assess the effect of LGB321 on proliferation, cells were measured at seeding time) is the 0% growth (100% inhi- seeded in 96-well tissue culture plates followed by addition bition). The crossing point defines the concentration at of compound that had been serially diluted to achieve a which growth is inhibited by 50% and is reported as GI50 final concentration range of 10 mmol/L to 2 nmol/L in 0.1% in this report. dimethyl sulfoxide (DMSO). After addition of LGB321 to cells, assay plates were returned to a humidified CO2 incu- Phosphoprotein assays bator (37 C; 5% CO2) for 3 days. To determine KMS-11.luc Commercial electrochemiluminescence (ECL) assay kits cell growth, 100 mL per well of reconstituted CellTiter-Glo from Meso Scale Discovery (MSD) were used to quantify the reagent was added to the cell assay plates. Assay plates were effects of LGB321 on the levels of phosphorylated S6RP and then sealed and shaken on a DELFIA (PerkinElmer) plate- BAD in KMS-11.luc cells. shaker for 10 minutes at 400 to 600 rpm. Plates were then For in vitro assays, KMS-11.luc cells were seeded in 96-well read on either a Microbeta Trilux (PerkinElmer) or Spectro- tissue culture plates, and then LGB321 was serially diluted Max L (Molecular Devices) luminometer. Cell growth was and added to cell plates to achieve a final concentration determined by comparing assay signals of LGB321 treated range of 10 mmol/L to 2 nmol/L in 0.1% DMSO and cells with the control conditions of untreated cells (defining incubated with cells for 1 hour at 37C. Cells were pelleted 0% growth inhibition) or cells treated with KI-1, a potent by centrifugation for 7 minutes at 1,500 rpm, media was nonspecific cytotoxic kinase inhibitor (defining 100% gently aspirated, and MSD lysis buffer added. Cells were growth inhibition). lysed by placing plates on a DELFIA (PerkinElmer) plate

OF4 Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research

Pan-PIM Kinase Inhibition in Hematologic Cancers

shaker at 4C and shaking the plates for 30 minutes at 600 Results rpm. Comprehensive analysis of PIM kinases mRNA in vivo For assays, MDS lysis buffer (MSD) was added to Expression frozen pulverized tumor samples on ice and homogenates Elevated expression of PIM kinases and their role in were prepared using the MagNA Lyser bead instrument cancer progression have been extensively reported in the (Roche Applied Science) by disrupting the samples with literature (4). However, a comprehensive evaluation of their four cycles of 6,000 rpm for 30 seconds at 4 C. Supernatants expression in a large number of samples across normal and were created following centrifugation at 11,000 rpm for 15 cancer tissue types has not been reported. Given that PIMs minutes at 4 C, and protein concentration determined are constitutively active kinases (3), we reasoned that high using the BCA Protein Assay Kit according to the manufac- mRNA expression could be used to identify cancers in which turer’s instructions (Pierce Chemical Company). For both PIM kinases are active. We evaluated datasets derived from assays, samples were transferred to ECL assay plates previ- Affymetrix human genome U133 arrays from public depos- ously blocked with 3% BSA, sealed, and incubated at 4 C itories for PIM kinases expression. All primary data were overnight while undergoing gentle shaking on a DELFIA normalized to allow direct comparison of expression across (PerkinElmer) plate shaker. After overnight incubation, all tissues (Materials and Methods). Expression of PIM1, assay plates were processed according to the manufacturer’s PIM2, and PIM3 was examined in normal and malignant instructions. samples derived from 17 different tissue types (Supplemen- LGB321 in vivo studies tary Fig. S1). Consistent with their known roles in cytokine All studies were done in an Association for Assessment signaling in hematopoiesis (2), all three PIMs are expressed and Accreditation of Laboratory Animal Care-accredited at higher levels in hematologic samples as compared with animal facility and in compliance with the ILAR Guide for other tissues (Fig. 1 and Supplementary. Fig. S1). Increased theCareandUseofLaboratoryAnimals.Scid/bgfemale expression of PIMs in most cancer types over their normal mice (10–12-week-old mice weighing about 20 g each; tissues counterpart is rather modest on average, suggesting CharlesRiverLaboratories)werehousedupto5animals that PIM kinases expression is primarily hematologic line- per cage in clear polycarbonate microisolator cages with a age-specific. 12-hour light, 12-hour dark cycle at temperatures Interestingly, across hematologic malignancies various between 70Fto80F, and 30% to 70% relative humidity. PIM isoforms are expressed at higher levels in acute lym- Food (Purina rodent chow pellets) and water were pro- phoblastic leukemia (ALL), AML, multiple myeloma, or vided ad libitum. DLBCL-NHL samples, relative to samples of liver, lung, For subcutaneous tumor models, cells were harvested at pancreas, prostate, and stomach cancers (Fig. 1). PIM2 80% to 90% confluency, washed, and resuspended in cold expression in multiple myeloma seems to be significantly þ Dulbecco’s Phosphate-Buffered Saline (without Ca2 or higher than all other tissues examined, including normal þ Mg2 ; CellGro) at a concentration of 5 107 cells/mL, bone marrow, whereas PIM1 expression is higher in AML, mixed with an equal volume of Matrigel (Becton-Dickin- ALL, and DLBCL than in multiple myeloma (Fig. 1). In son) and then 0.2 mL (5 106 cells) was implanted addition to the functional similarities as oncogenes (7, 8), subcutaneously into the right flank of female Scid/bg mice. this observation suggested that in order for a small-mole- Tumor volume was measured in two dimensions using cule inhibitor to have broad clinical utility the effective inhibition of all three PIM kinases (Pan-PIM) would be digital calipers and calculated as ðlength width2Þp=6. required. Because we had observed that the multiple mye- For pharmacodynamic and pharmacokinetic studies, loma KMS-11.luc cells are dependent on PIM2 for prolif- animals were enrolled on study when mean tumor vol- eration (14) and because PIM2 inhibition is the most ume reached 250 to 400 mm3. For efficacy studies, ani- challenging in the cellular context (as described in the next mals were randomized into groups when tumor volume section), we chose this cell line to drive the development of reached 200 to 250 mm3. Tumor volume and body potent Pan-PIM inhibitors in cells. weights were captured and stored by StudyDirector software (StudyLog). LGB321 was formulated for oral administration in 50 Biochemical activity and selectivity of the Pan-PIM mmol/L acetate buffer, pH4. The concentration of LGB321 inhibitor LGB321 in plasma was determined following extraction in acetoni- We reasoned that an ATP-competitive inhibitor was most trile using liquid chromatography and tandem mass spec- likely to inhibit all three PIM kinases, because the ATP- troscopy. Cytarabine (Hospira) was diluted in bacteriostatic binding pocket is highly conserved among the three PIM water and prepared fresh daily. kinases and has some unique features relative to the ATP pocket for other kinases (3). The development of a Statistical analysis cell-active ATP-competitive inhibitor for PIM2 was partic- For in vivo studies, statistical significance of differences in ularly challenging, given its low ATP Km (4 mmol/L; Fig. 2B) tumor volume was determined by a one-way ANOVA, and as compared with the Km for PIM1 or PIM3 (400 and 40 pair-wise comparisons were made by the uncorrected Fisher mmol/L, respectively; Fig. 2B). Thus, the desired inhibitor LSD posttest (GraphPad Prism software). needed to be potent enough to compete with the large

www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 OF5

Garcia et al.

fi fi A Table 1. LGB321 Biochemical speci city pro le Structure of LGB321 Kinase IC (mmol/L) OH 50 PIM2 <0.003 H2N GSK3b 4.4 F F PKN1 4.4 F PKA 6.7 N N H S6K 6.8 N PKCa 7.0 PKN2 7.2 O PKCt 7.7 N ROCK2 9.4 B Biochemical potency of LGB321 in PIM kinases NOTE: Kinases with IC50 > 10 mmol/L: cABL(T315I), cABL, ALK, AURORA-A, BTK, CDK1B, CDK2A, CDK4D1, CK1, ATP Km LGB321 Ki COT1, CSK, CaMK2, ERK2, EPHA4, EPHB4, FAK, FGFR1, PIM1 400 µmol/L 1.0 pmol/L FGFR2, FGFR3, FGFR4, FGFR3(K650E), FLT3, FYN, HCK, µ PIM2 4 mol/L 2.1 pmol/L HER1, HER2, HER4, IGF1R, INS1R, IRAK4, JAK1,JAK2, µ JAK3, JNK2, JNK3, KDR, cKIT, LCK, LYN, cMET, MK2, PIM3 40 mol/L 0.8 pmol/L MK5, MNK1, MNK2, PAK2, PDGFRa, PDK1, PI3Ka, PI3Kb, PI3Kd, PI3Kg, PI4Kb, PKBa, PLK1, RET, RON, cSRC, SYK, Figure 2. LGB321 structure and biochemical properties. A, chemical mTOR, TYK2, VPS34, WNK1, YES, ZAP70, p38a, p38g. structure of LGB321. B, summary of the ATP Km and LGB321 inhibition constant (Ki) determined as described in Materials and Methods. We further evaluated the activity of LGB321 at the cellular level on two of the potential off-targets. First, we monitored excess of cellular ATP (in the range of 1–10 mmol/L; ref. 19), the activity of LGB321 on GSK3b, the most potent off-target about 250- to 2,500-fold higher than the Km for ATP of kinase identified in the biochemical assay (Table 1). As seen PIM2. We developed a series of highly potent and selective in Supplemental Fig. S4 early compounds of the LGB321 inhibitors of all three PIM kinases (20) and selected LGB321 series showed cellular inhibition of GSK3b, as demonstrat- (Fig. 2A) as a tool compound with such characteristics. The ed by the stabilization of b-catenin. However, compound inhibition constant (Ki) for LGB321 was determined to be optimization increased the biochemical selectivity and in the single-digit picomolar (pmol/L) range for each of the resulted in complete lack of activity of LGB321 on GSK3b three PIM kinases (Fig. 2B). Several groups have developed in cells (Supplementary. Fig. 4). We next evaluated the inhibitors to the PIM kinases in both academic and industry activity of LGB321 on EGFR, as it was identified as the most settings (10–13); however, to our knowledge LGB321 is the potent off-target in the KINOMESCAN-binding displace- most potent compound so far described. When directly ment assay. Despite this result, we found no evidence of compared with two other PIM inhibitors tested in clinical inhibitory activity in EGF signaling in the cellular context trials, it is clearly more potent (Supplementary Fig. S2; (Supplementary. Fig. S5). Together these results reinforce ref. 20). that LGB321 is a highly potent and selective Pan-PIM The selectivity of LGB321 was first determined in bio- inhibitor. chemical assays of a panel of seven lipid kinases and 68 diverse protein kinases that included PIM2 (Table 1). In this Cellular activity of LGB321 panel, only PIM2 was significantly inhibited by LGB321 The cellular activity of LGB321 was evaluated in KMS-11. with an IC50 of <0.003 mmol/L, the lowest sensitivity range luc cells, a KMS-11 clone expressing firefly luciferase that we for the assay. Although biochemical potency in the range of have demonstrated previously to be dependent on PIM2 4to10mmol/L was demonstrated against eight other kinase activity (14). Western blotting (Fig. 3A) was used to kinases in this assay, these IC50 represent a greater than demonstrate that LGB321 inhibited the phosphorylation of 6 10 -fold differential relative to the Ki on all three PIM BAD at Ser-112 (a direct PIM kinase substrate; refs. 21–23) kinases. The biochemical IC50 for all other kinases tested and two proteins downstream of the mTOR-C1 complex in this panel was >10 mmol/L (Table 1). We further evalu- (S6K at Thr-389 and its substrate S6RP at Ser-235/6; ref. 24) ated the selectivity of LGB321 using the KINOMESCAN- in a concentration-dependent manner. The effect of binding displacement assay (Supplementary. Fig. S3; LGB321 in phosphorylation of S6K or S6RP is not due to ref. 20). The results demonstrate that LGB321 has a high an off-target effect on mTOR, because the compound was selectivity score with activity against only two other kinases inactive in the mTOR biochemical assay shown in Table 1 (EGFR and ERK8) in this assay, in addition to the PIM and did not inhibit the phosphorylation of S6K or S6RP in kinases (Supplementary Fig. S3; ref. 20). 293A/TSC2-null cells with constitutively active mTOR (14).

OF6 Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research

Pan-PIM Kinase Inhibition in Hematologic Cancers

Activity of LGB321 in a large panel of cancer cell lines AB Having established the potency, selectivity, and cellular LGB321 (µmol/L) activity of LGB321, we proceeded to use it to test the 100 0 0.1 1.0 hypothesis that PIM inhibition would have the greatest 0.33 0.033

389 pThr -S6K 80 impact on cell lines derived from hematologic lineages in which PIM kinases are most highly expressed. To do this, we Total-S6K took advantage of our high-throughput compound screen 60 pS6RP(S235/6) Inhibition pSer235/6-S6RP pS6RP(S240/4) Inhibition of more than 500 cell lines from the CCLE (18). It should be pBAD(S112) Inhibition Total-S6RP 40 Proliferation noted that in addition to test compounds of interest, many

pSer112-BAD Percent response other well-characterized inhibitors and cancer drugs have 20 been included in the CCLE screens, including panobinostat, Total-BAD erlotinib, and PLX4720 (18). These inhibitors serve as Actin 0 0.001 0.01 0.1 1 10 controls for specificity by showing differential activity in Pim2 Concentration LGB321 (µmol/L) cell lines of diverse cancer lineages or genetic backgrounds (18). LGB321 has been tested in this screen on three C different occasions, and in each case with similar results Substrate EC (µmol/L) N 50 (data not shown). For simplicity, the data from a single pBAD (S112) 0.010 ± 0.006 16 screen are presented here to demonstrate the pattern of 235/6 ± pS6RP (S ) 0.026 0.013 4 LGB321 activity. As is apparent in Fig. 4A, LGB321 displays 240/4 ± pS6RP (S ) 0.038 0.027 17 very limited activity in most cell lines derived from solid Proliferation 0.017 ± 0.011 26 tumors, including breast, central nervous system, kidney, large intestine, liver, lung, ovarian, pancreas, prostate, and Figure 3. Effect of LGB321 on PIM2 target modulation and proliferation stomach cancer cell lines (Fig. 4A). The limited activity in KMS-11.luc cells. A, Western blot analysis of KMS-11.luc cells observed in some lung cancer cell lines was not due to treated with a dose–response of LGB321 for 3 hours. B, quantitative potential activity against EGFR because LGB321 does not analysis of the effects of LGB321 on KMS-11.luc proliferation and inhibit EGFR signaling in cells (Supplementary Fig. S5). phosphorylation of S6RP and BAD; cells treated with a dose–response of LGB321 were tested in 72-hour proliferation assays as well as This was further supported by the fact that the lung cell lines quantitative ECL assays as described in Materials and Methods. tested showed differential sensitivity to LGB321 and erlo- C, summary of the quantitative cellular activities on LGB321 in tinib, a potent and specific EGFR inhibitor (Supplemen- KMS-11.luc cells. tary Table S1). In general, the lack of activity of LGB321 in cell lines derived from solid tumors, including cancer Thus, PIM kinases likely act upstream of mTOR to regulate indications for which evidence of roles for PIM kinases its activity, a finding that agrees with our recent data dem- have been reported, was somewhat surprising. A possible onstrating that PIM2 regulates mTOR through phosphor- explanation for this observation is suggested by the recent ylation of TSC2 in the tuberous sclerosis complex (14). report that PIM kinase inhibition may result in increased To better understand the relationship between the bio- expression of several tyrosine kinase receptors in prostate chemical and cellular potency of LGB321, we used quan- cancer cells (25), which activates the mitogen-activated titative Meso Scale assays designed to measure levels of protein kinase and PI3K/AKT pathway as the main drivers phospho-BAD (pBAD) and phospho-S6RP (pS6RP). Sim- of proliferation in these cells. Nevertheless, our results ilar to the Western blot analysis results, these assays confirm withahighlypotentandselective inhibitor suggest that in that LGB321 inhibits phosphorylation of both BAD and solid tumors PIM kinases are not the primary driver of S6RP (at both the Ser-235/6 and Ser-240/6 sites, respec- proliferation. tively) in a concentration-dependent manner in KMS-11.luc To further explore the LGB321 activity in hematologic cells (Fig. 3B). In parallel experiments, we also determined malignancies, we tested it in a screen that was adapted to the effect of LGB321 on the proliferation of KMS-11.luc cells evaluate the sensitivity of compounds on an extended panel using CellTiter-GLO assays. The potency of LGB321 in of hematologic cell lines (Fig. 4B; Supplementary. Table modulating PIM signaling correlated well with the inhib- S2). In this screen, cell lines from ALL, AML, multiple itory effects on KMS-11.luc cell proliferation (Fig. 3B and myeloma, and B-cell NHL were represented with more than C). We obtained similar results in KMS-26, KMS-34, and 18 cell lines each. Multiple myeloma cells seem to be the H929 multiple myeloma cells, in which LGB321 inhibition most broadly sensitive to LGB321 with 14 of the 18 cell lines of proliferation and PIM signaling was observed (14). tested having GI50 below 1 mmol/L (Fig. 4B; Supplementary Interestingly, the inhibition of pBAD by LGB321 in these Table S2). Highly sensitive to LGB321 was also identified in multiple myeloma cell lines did not correlate with signif- ALL, AML, CML, and NHL cell lines. However, the response icant induction of PARP cleavage (14). Taken together, our was less broad than in the multiple myeloma panel, with 4 results demonstrate that LGB321 is a potent and selective of 21 ALL, 15 of 26 AML, and 10 of 27 B-cell NHL cell lines Pan-PIM inhibitor capable of inhibiting PIM kinase signal- showing sensitivity to LGB321 with GI50 below 1 mmol/L. In ing and proliferation in PIM2-dependent multiple myelo- addition, activity was also observed in Hodgkin lymphoma, ma cell lines. CML, and T-cell NHL cell lines. However, the number of cell

www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 OF7

Garcia et al.

A Breast Hematopoietic CNS Liver Lung Ovary Skin

0.5

-0.5 ( µ mol/L)

50 -1.0

of Gl -1.5 10

-2.0 Figure 4. LGB321 screen in CCLE. Log A, cell lines are grouped and color -2.5 coded according to the primary site Cell lines of the tumor from which they were derived. To better demonstrate the differences in Autonomic ganglia Hematopoietic Ovary Stomach sensitivity across the cell lines, the Biliary tract Kidney Pancreas Thyroid directionality of bars in the graph is based on a concentration of 1 Bone Large intestine Pleura Upper aerodigestive mmol/L (log 0), with cell lines in Breast Liver Prostate Urinary tract which 50% growth inhibition was achieved at lower concentrations CNS Lung Skin (Not-specified) than 1 mmol/L dropping down, Endometrium Esophagus Soft tissue and those in which it was higher than 1 mmol/L rising up. Hematopoietic cancers are the most sensitive to PIM inhibition. B, LGB321 screen in an expanded B ALL AML CML MM NHL panel of cell lines from hematologic 1 (B Cell) malignancies. LGB321 is most broadly effective in MM. In AML, a subset of cell lines are very 0.5 sensitive to LGB321. Sensitivity in ALL, CML, Hodgkin lymphoma, and NHL cell lines was also mol/L)

µ observed. ( µ 0 50

of GI -0.5 10 NHL

Log (T Cell) -1

-1.5

Hodgkin

lines representing these diseases is rather low. Furthermore, Activity of LGB321 in vivo the genetic alterations frequently observed in these diseases We have reported elsewhere the antitumor activity of are represented in only a few cell lines. Thus, further LGB321 in the multiple myeloma KMS-11.luc xenograft exploration will be required to understand the role of PIM model, together with a detailed characterization of its kinases in these malignancies and their diverse genetic mechanism of action in multiple myeloma cells (14). Here, backgrounds. The broad distribution of LGB321 sensitivity we focus on the characterization of the LGB321 activity in among hematologic cell lines, including both lymphoid an AML xenograft model. Among the sensitive AML cell and myeloid lineages, suggests that PIM kinase inhibition lines tested in the expanded hematologic cell line panel may have a broad application on the treatment of these (Fig. 4B; Supplementary Table S2), KG-1 cells were chosen malignancies. as a model that readily grows in vivo. We ﬁrst veriﬁed that

OF8 Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research

Pan-PIM Kinase Inhibition in Hematologic Cancers

Figure 5. LGB321 activity in AML xenografts and demonstration of in vivo synergy with cytarabine. A, modulation in vitro of pBAD and mTOR signaling in KG-1 cells. B, single-dose pharmacodynamic/ pharmacokinetic of LGB321 in mice bearing subcutaneous KG-1 tumors. C, LGB321 efficacy as single agent and in combination with cytarabine. LGB321was dosed by oral gavage with vehicle, LGB321 at 30, 100 mg/kg once daily (QD), cytarabine at 100 mg/kg QD by intraperitoneal injection, or with a combination of cytarabine and 30 or 100 mg/kg LGB321 (n ¼ 9/group). Although 30 mg/kg QD of LGB321 resulted in significant tumor growth inhibition (, P < 0.05), 100 mg/kg QD achieved the greater effect of partial regression. In contrast, cytarabine has no effect in this model as a single agent. However, when combined with 30 mg/kg QD of LGB321, a synergistic effect was achieved resulting in tumor regression (P < 0.05). When cytarabine was combined with 100 mg/kg LGB321, this also resulted in increased tumor regression, though this result was not statistically significant compared with the 100 mg/kg LGB321 group alone. The combination of LGB321 and cytarabine led to significant body weight loss after 5 successive days of dose, and the combination groups were granted a 2-day dosing holiday before administration continued.

KG-1 cells were indeed sensitive to LGB321 in several (Fig. 5A). Having established in vitro the responses of independent CellTiter-GLO proliferation assays (GI50 of KG-1 cells to PIM inhibition, we proceeded to evaluated 0.08 0.07 mmol/L; N ¼ 7). We then tested whether PIM in vivo the LGB321 pharmacokinetic–pharmacodynamic inhibition in KG-1 cells resulted in modulation of pBAD relationship in KG-1 subcutaneous tumors (Fig. 5B). and the mTOR signaling pathway and found that indeed Following a single oral dose of LGB321 (30 or 100 LGB321 effectively inhibited both signaling pathways mg/kg), at the indicated time points plasma and tumor

www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 OF9

Garcia et al.

samples were collected for pharmacokinetics and phar- LGB321, a potent and selective inhibitor of the PIM kinase macodynamics analysis, respectively. PIM kinase inhibi- family members capable of inhibiting PIM2 at the high tion was determined by assessing the modulation of cellular concentrations of ATP (20). Because LGB321 is a pS6RP and pBAD in tumor lysate using the quantitative picomolar inhibitor of all three PIM family members, we Meso Scale assay and the results were expressed as a ratio carefully evaluated its selectivity against other kinases in of their phosphorylated to unphosphorylated forms (Fig. both biochemical and cellular contexts. The selectivity was 5B). A dose-dependent increase in the plasma concentra- tested biochemically in a panel of 75 kinases (Table 1) and tion of LGB321 was observed (Fig. 5B). At the 100 mg/kg by binding displacement assays in a panel of 386 kinases dose, the plasma concentration was maintained at higher (Supplementary Fig. S3; ref. 20). Furthermore, cellular levels through 24 hours, whereas at the 30 mg/kg dose a assays for the closest off-target kinase in each of these panels gradual reduction in the plasma concentration occurred. (Supplementary Figs. S4 and S5) were used to demonstrate The modulation of pS6RP in tumors was also dose- that LGB321 was inactive on these kinases at concentrations dependent with more sustained inhibition at 100 mg/kg well above the on-target activities on PIM2-dependent cells but only transient inhibition at 30 mg/kg. In contrast, (Fig. 3). Selectivity was further evident by the almost exclu- both doses achieved equally significant and sustained sive activity of LGB321 in hematologic cell lines among pBad inhibition through 24 hours. This observation more than 500 cell lines in the CCLE. raises the question of which target modulation marker LGB321 as single agent was well-tolerated in vivo after would better predict the in vivo efficacy of LGB321. multiple doses, a finding predicted by the viability and To address this question, we tested the same 30 and 100 fertility of mice with knockout of the three PIM kinase mg/kg doses of LGB321 in a daily regimen of oral dosing in genes (27). LGB321 can be administered orally to reach an 11-day efficacy study. At the 30 mg/kg daily regimen of plasma exposures that allowed us to evaluate the relation- LGB321, we observed near stasis with only minor increase ship between pharmacokinetics, pharmacodynamics, and in tumor volume relative to the vehicle control (Fig. 5B). In efficacy of PIM inhibition in xenograft models. Although contrast, slight tumor regression was observed with the 100 oral administration with LGB321 leads to a dose-dependent mg/kg daily regimen. These data establish that the extent increase in plasma exposure, the increased exposure was and duration of pS6RP, and not pBad, modulation corre- achieved through extended plasma drug concentrations, lates with efficacy in this AML model, a finding in agreement rather than successive increases in peak plasma concentra- with our previous data in multiple myeloma models (14). tion, as exhibited by differences between the 30 and 100 We have also recently reported similar in vivo activity of mg/kg doses (Fig. 5B). The pharmacokinetic properties of LGB321 in the AML EOL1 xenograft model (20). Here, we LGB321 at higher doses are somewhat advantageous, as the tested the combination of LGB321 with the nucleoside extended exposure is concomitant with sustained target analog cytarabine, a standard-of-care in the clinical treat- inhibition as evidenced by pS6RP inhibition. The maximal ment of AML. KG-1 tumors did not exhibit a significant in vivo effect was slight tumor regression at the 100 mg/kg/d response to cytarabine alone when delivered at 100 mg/kg regimen in the AML KG-1 model (Fig. 5C) and tumor stasis daily, a dose that results in clinically relevant exposures in the KMS-11.luc (14) and the EOL-1 models (20). As this (26). When 30 mg/kg LGB321 daily dose was combined dose is near the maximal tolerated dose, we could not assess with 100 mg/kg daily dose of cytarabine, a synergistic effect whether LGB321 would achieve tumor regression at higher resulting in slight regression was achieved (Fig. 5B). When exposures. Nevertheless, stasis or slight regression are con- combined with the higher dose of LGB321, the synergistic sistent with our results that LGB321 leads to inhibition of effect was not statistically significant, given that regression cell proliferation and not to apoptosis as assessed by PARP was achieved with 100 mg/kg LGB321 alone. However, the cleavage in the KMS-11.luc model (14). In the KG-1 model, combination of cytarabine with LGB321 induced signifi- tumor regression of approximately 50% was observed when cant body weight loss following the fifth day of adminis- LGB321 was coadministered with cytarabine, a nucleoside tration (not shown), which required dosing to be halted analog that is integral to the current standards of care in only in the combination arms for 2 days before resuming AML (28). Interestingly, in this model cytarabine alone had treatment. These results demonstrate the efficacy of LGB321 no effect, highlighting the suggestion that the combined both as a single agent as well as in combination with used of this agent and PIM inhibitors could enhance the cyatarabine, even in a model that is refractory to this clinical response (29). standard of care as a single agent. The higher level of PIM mRNA expression in hematologic tissues is consistent with a distinct role for PIM kinases in Discussion hematologic cancers. We demonstrated that PIM kinase Here, we demonstrated that each of the three PIM kinase inhibition has an antiproliferative effect primarily in hema- family members is most highly expressed in at least one tologic cell lines, with very limited activity in cell lines from subtype of hematologic cancer. This observation coupled solid tumors. Although the role of PIM kinase in some solid with the fact that PIM family members can substitute for tumors has been extensively described in the literature each other to generate lymphomas (6, 7) indicates that to (2, 4), our data suggest that PIM kinases play a more effectively treat hematologic cancers, an inhibitor of all significant role in promoting proliferation in hematologic three isoforms is required. Furthermore, we describe malignancies than in solid tumors. Interestingly, PIM

OF10 Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research

Pan-PIM Kinase Inhibition in Hematologic Cancers

kinases were identified as oncogenes by insertional muta- Authors' Contributions genesis screens in lymphomas from murine leukemia virus– Conception and design: P.D. Garcia, J.L. Langowski, C. Fanton, T. Zavor- otinskaya, X.-H. Niu, J. Lan, M. Lindvall, J. Holash, M.T. Burger infected mice (8, 30), but not in tumors induced by the Development of methodology: P.D. Garcia, J.L. Langowski, M. Chen, J. mouse mammary tumor virus (31). These observations Castillo, M. Ison, Y. Dai, X.-H. Niu, S. Basham, J. Chan, J. Yu, M. Doyle, P. suggest that PIM kinases, in addition to being specifically Feucht, C. Fritsch, E. Pfister, J.A. Aycinena Acquisition of data (provided animals, acquired and managed patients, expressed in the hematologic lineage, are hematologic provided facilities, etc.): J.L. Langowski, Y. Wang, C. Fanton, T. Zavor- lineage-specific oncogenes. Our results that the inhibitory otinskaya, J. Lu, X.-H. Niu, S. Basham, P. Feucht, R. Warne, E. Pfister, P. Drueckes, J. Trappe, C. Wilson, C. Bellamacina, J.A. Aycinena, R. Zangl activity of LGB321 is observed primarily in cell lines of Analysis and interpretation of data (e.g., statistical analysis, biosta- hematologic lineage further strengthen the notion that PIM tistics, computational analysis): P.D. Garcia, J.L. Langowski, M. Chen, C. kinases play a lineage-related role in promoting prolifera- Fanton, T. Zavorotinskaya, X.-H. Niu, S. Basham, J. Yu, P. Feucht, C. Fritsch, A. Kauffmann, E. Pfister, P. Drueckes, J. Trappe, C. Wilson, J.A. Aycinena, R. tion of some hematologic cancers and suggest that poten- Zang, J. Holash tially single-agent activity may be observed in these cancers. Writing, review, and/or revision of the manuscript: P.D. Garcia, J.L. In aggregate, our results strongly suggest that the use of Langowski, C. Fanton, X.-H. Niu, S. Basham, M. Doyle, P. Feucht, J. Holash Administrative, technical, or material support (i.e., reporting or orga- potent and selective pan-PIM inhibitors, either as single nizing data, constructing databases): J. Castillo, M. Ison, Y. Dai, X.-H. agent or in combination with other agents, will be very Niu, J. Narberes, T. Tsang, W. Han, G. Nishiguchi useful for the treatment of hematologic malignancies in Study supervision: P.D. Garcia, T. Zavorotinskaya, X.-H. Niu, S. Basham general, and in multiple myeloma and AML in particular. To Acknowledgments test this hypothesis in humans, we have initiated the phase I The authors thank Dr. Suzanne Trudel at the UHN for generously clinical testing of our development candidate LGH447 in providing the KMS-11.luc cell line and Dr. James D. Griffin at the Dana- Farber Cancer Institutes (Boston, MA) for his helpful advice. The authors also relapsed and/or refractory multiple myeloma. thank Gary Vanasse, Chuck Voliva, Emma Lees, and William Sellers for their support and critical reading of the article. The costs of publication of this article were defrayed in part by the fl payment of page charges. This article must therefore be hereby marked Disclosure of Potential Con icts of Interest advertisement P.D. Garcia has ownership interest (including patents) in Novartis Shares. in accordance with 18 U.S.C. Section 1734 solely to indicate J.L. Langowski is an investigator in Novartis. J. Lan is a research investigator this fact. III in Novartis. M. Lindvall has ownership interest (including patents) in Novartis. J. Holash has ownership interest (including patents) in Novartis Received July 29, 2013; revised December 6, 2013; accepted January 7, stock. No potential conflicts of interest were disclosed by the other authors. 2014; published OnlineFirst January 28, 2014.

References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer PIM-2, and PIM-3 protein kinases. Bioorg Med Chem Lett 2012;22: J Clin 2012;62:10–29. 4599–604. 2. BraultL,Gasser C, Bracher F, Huber K, Knapp S, Schwaller J. PIM serine/ 12. Morwick T. Pim kinase inhibitors: a survey of the patent literature. threonine kinases in the pathogenesis and therapy of hematologic Expert Opin Ther Pat 2010;20:193–212. malignancies and solid cancers. Haematologica 2010;95:1004–15. 13. Narlik-Grassow M, Blanco-Aparicio C, Carnero A. The PIM family of 3. QianKC,WangL,HickeyER,StudtsJ,BarringerK,PengC,etal. serine/threonine kinases in cancer. Med Res Rev 2013;34:136–59. Structural basis of constitutive activity and a unique nucleotide 14. Lu J, Zavorotinskaya T, Dai Y, Niu XN, Castillo J, Sim J, et al. Pim2 is binding mode of human Pim-1 Kinase. J Biol Chem 2005;280: required for maintaining multiple myeloma cell growth through mod- 6130–7. ulating TSC2 phosphorylation. Blood 2013;122:1610–20. 4. Nawijn MC, Alendar A, Berns A. For better or for worse: the role of Pim 15. Dai M, Wang P, Boyd AD, Kostov B, Athey B, Jones EG, et al. Evolving oncogenes in tumorigenesis. Nat Rev Cancer 2011;11:23–34. gene/transcript definitions significantly alter the interpretation of Gen- 5. Cuypers HT, Selten G, Quint W, Zijlstra M, Maandag ER, Boelens W, eChip data. Nucleic Acids Res 2005;33:e175. et al. Murine leukemia virus-induced T-cell lymphomagenesis: inte- 16. Harbron C, Chang KM, South MC. RefPlus: an R package extending gration of proviruses in a distinct chromosomal region. Cell 1984; the RMA Algorithm. Bioinformatics 2007;23:2493–4. 37:141–50. 17. Manley PW, Drueckes P, Fendrich G, Furet P, Liebetanz J, Martiny- 6. Meeker TC, Nagarajan L, ar-Rushdi A, Croce CM. Cloning and char- Baron G, et al. Extended kinase profile and properties of the protein acterization of the human PIM-1 gene: a putative oncogene related to kinase inhibitor nilotinib. Biochim Biophys Acta 2010;1804:445–53. the protein kinases. J Cell Biochem 1987;35:105–12. 18. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim 7. van der Lugt NM, Domen J, Verhoeven E, Linders K, van der Gulden H, S, et al. The cancer cell line encyclopedia enables predictive modelling Allen J, et al. Proviral tagging in E mu-myc transgenic mice lacking the of anticancer drug sensitivity. Nature 2012;483:603–7. Pim-1 proto-oncogene leads to compensatory activation of Pim-2. 19. Beis I, Newsholme EA. The contents of adenine nucleotides, phos- EMBO J 1995;14:2536–44. phagens, and some glycolytic intermediates in resting muscles from 8. Mikkers H, Allen J, Knipscheer P, Romeyn L, Hart A, Vink E, et al. High- vertebrates and invertebrates. Biochem J 1975;152:23–32. throughput retroviral tagging to identify components of specific sig- 20. Burger MT, Han W, Lan J, Nishigushi G, Bellamacina C, Lindval M, et al. naling pathways in cancer. Nat Genet 2002;32:153–9. Structure guided optimization, in vitro activity, and in vivo activity of 9. van Lohuizen M, Verbeek S, Krimpenfort P, Domen J, Saris C, Radasz- Pan-PIM kinase inhibitors. ACS Med Chem 2013;4:1193–7. kiewicz T, et al. Predisposition to lymphomagenesis in pim-1 trans- 21. Macdonald A, Campbell DG, Toth R, McLauchlan H, Hastie CJ, Arthur genic mice: cooperation with c-myc and N-myc in murine leukemia JS. Pim kinases phosphorylate multiple sites on Bad and promote 14- virus-induced tumors. Cell 1989;56:673–82. 3-3 binding and dissociation from Bcl-XL. BMC Cell Biol 2006;7:1. 10. Anizon F, Shtil AA, Danilenko VN, Moreau P. Fighting tumor cell 22. Yan B, Zemskova M, Holder S, Chin V, Kraft A, Koskinen PJ, et al. The survival: advances in the design and evaluation of Pim inhibitors. Curr PIM-2 kinase phosphorylates BAD on serine 112 and reverses BAD- Med Chem 2010;17:4114–33. induced cell death. J Biol Chem 2003;278:45358–67. 11. Dakin LA, Block MH, Chen H, et al. Discovery of novel benzylidene-1,3- 23. Aho TL, Sandholm J, Peltola KJ, Mankonen HP, Lilly M, Koskinen PJ. thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein

www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 OF11

Garcia et al.

by phosphorylating it on the Ser112 gatekeeper site. FEBS Lett 2004; 28. Roboz GJ. Novel approaches to the treatment of acute myeloid 571:43–9. leukemia. Hematology Am Soc Hematol Educ Program 2011; 24. Mamane Y, Petroulakis E, LeBacquer O, Sonenberg N. mTOR, trans- 2011:43–50. lation initiation, and cancer. Oncogene 2006;25:6416–22. 29. Kelly KR, Espitia CM, Taverna P, Choy G, Padmanabhan S, 25. Cen B, Mahajan S, Wang W, Kraft AS. Elevation of receptor tyrosine Nawrocki ST, et al. Targeting PIM kinase activity significantly aug- kinases by small molecule AKT inhibitors in prostate cancer is medi- ments the efficacy of cytarabine. Br J Haematol 2012;156: ated by Pim-1. Cancer Res 2013;73:3402–11. 129–32. 26. Zuber J, Radtke I, Pardee TS, Zhao Z, Rappaport AR, Luo W, et al. 30. Sauvageau M, Miller M, Lemieux S, Lessard J, Hebert J, Sauvageau G. Mouse models of human AML accurately predict chemotherapy Quantitative expression profiling guided by common retroviral inser- response. Genes Dev 2009;23:877–89. tion sites reveals novel and cell type specific cancer genes in leukemia. 27. Mikkers H, Nawijn M, Allen J, Brouwers C, Verhoeven E, Jonkers J, Blood 2008;111:790–9. et al. Mice deficient for all PIM kinases display reduced body size and 31. Theodorou V, Kimm MA, Boer M, Wessels L, Theelen W, Jonkers J, et al. impaired responses to hematopoietic growth factors. Mol Cell Biol MMTV insertional mutagenesis identifies genes, gene families, and path- 2004;24:6104–15. ways involved in mammary cancer. Nat Genet 2007;39:759–69.

OF12 Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research

Pan-PIM Kinase Inhibition Provides a Novel Therapy for Treating Hematologic Cancers

Pablo D. Garcia, John L. Langowski, Yingyun Wang, et al.

Clin Cancer Res Published OnlineFirst January 28, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-13-2062

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2014/03/28/1078-0432.CCR-13-2062.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2014/03/13/1078-0432.CCR-13-2062. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.