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Cancer Therapy: Preclinical

Pan-PIM Kinase Inhibition Provides a Novel Therapy forTreating Hematologic Cancers

Pablo D. Garcia1, John L. Langowski1, Yingyun Wang1, Min Chen1, Joseph Castillo1, Christie Fanton1,Marjorie Ison1, Tatiana Zavorotinskaya1, YuminDai1, Jing Lu1, Xiao-HongNiu1, StephenBasham1, JulieChan1,Jianjun Yu1,Michael Doyle1, Paul Feucht1, RobertWarne1, 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

AbstractPurpose: PIMkinases have been shown to act as oncogenes inmice, with each familymember being able

to drive progressionofhematologic cancers. Consistentwith 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 prolifer-

ation, 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 antipro-

liferative activity and show that it synergizeswith cytarabine in anAMLxenograftmodel.Our results strongly

support the development of Pan-PIM inhibitors to treat hematologic malignancies. Clin Cancer Res; 20(7);

1–12. �2014 AACR.

IntroductionAs a group, hematologic cancers are the fourth most

common cancer type in the United States (1). In 2012, an

estimated 70,130 cases of non-Hodgkin lymphoma (NHL),21,700 cases of multiple myeloma, 16,060 cases of chroniclymphocytic leukemia (CLL), and 13,780 cases of acutemyelogenous leukemia (AML) were diagnosed (1). In spiteof considerable advanceswith novel therapeutics, includingmonoclonal antibodies, stem cell transplantation, and tar-geted therapies, the number of patients succumbing to thesediseases remains high, with more than 45,000 deaths esti-mated in 2012 (1).

In both hematologic cancers and solid tumors, therapiesthat target cancer drivers have been shown to be clinicallybeneficial. With this in mind, we identified the PIM familyof protooncogenes as a suitable target given that they arehighly expressed in hematologic malignancies. Althoughgene expression alone is not always a fair predictor of cancerrelevance, PIM kinases display a few unique features. Onceproperly folded, PIM kinases require no further posttrans-lational modifications for their activity (2); indeed, the

Authors' Affiliations: 1Oncology Disease Area Research; 2Global Discov-eryChemistry/Oncology and Exploratory Chemistry; 3MAPGroup; 4Chem-ical and Pharmaceutical Profiling Group, Novartis Institutes for BiomedicalResearch, Emeryville, California; 5Developmental Molecular Pathways,Novartis Institutes for Biomedical Research, Cambridge, Massachusetts;6Oncology Disease Area Research; and 7Center for Proteomic Chemistry,Novartis Institutes for Biomedical Research, Basel, Switzerland

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor:PabloD.Garcia, Novartis Institutes for BiomedicalResearch, 4560 Horton Street, Emeryville, CA 94608. Phone: 510-923-7680; Fax: 510-655-9910/510-923-2502; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-2062

�2014 American Association for Cancer Research.

ClinicalCancer

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crystal structure of PIM1 supports a model in which theunphosphorylated protein adopts an active conformation(3). In addition to transcriptional regulation, PIM kinasesare phosphorylated and autophosphorylated to regulatetheir stability (2). Thus, the cellular activity of each PIMkinase is primarily regulated by the balance of synthesis anddegradation of the protein itself (2), suggesting that expres-sion alone may be a useful predictor of where PIM kinasesare active.

The PIM kinases have roles in cell-cycle progression, cellsurvival, and tumorogenesis (4). PIM1 was originally dis-covered as an oncogene by frequent proximal proviralinsertions of murine leukemia virus in lymphomas ofinfected mice (5, 6). Similar unbiased insertional mutagen-esis screens were used to demonstrate the oncogenic poten-tial of PIM2 (7) and PIM3 (8) in PIM1 and PIM1/2 knock-out mice, respectively, suggesting that PIMs may havefunctional similarities in oncogenesis. These studies alsodemonstrated that PIM kinases in conjunction with c-Mycresult in synergistic induction of cancer (9).

Here, we describe LGB321, a potent and selective ATP-competitive small-molecule inhibitor of all three PIMkinases (Pan-PIM kinase inhibitor). LGB321 is uniquerelative to previously described PIM inhibitors (10–13), inthat it is active in PIM2-dependent cell lines (14), a kinasethat has proven difficult to inhibit in the cellular context.Consistentwith its activity on all three PIMkinases, LGB321inhibits proliferation of a number of cell lines derived fromdiverse hematologicmalignancies, includingmultiplemye-loma, AML, CML, and B-cell NHL. In vivo, the compound isorally available, demonstrates efficacy in tumor xenograftsand is well tolerated within the therapeutic exposure range

in mice. Taken together, these findings establish PIMkinases as an attractive pharmacologic target in cancertherapy.

Materials and MethodsSources of gene expression data

All gene expression microarray datasets were retrievedfrompublic depositories, includingGeneExpressionOmni-bus (http://www.ncbi.nlm.nih.gov/geo/) and ArrayExpress(http://www.ebi.ac.uk/arrayexpress/). Only human prima-ry tumor or normal tissue samples that were hybridized onAffymetrix human genome U133 Plus 2.0 arrays and wereobtained from patients that did not receive any treatmentbefore tissue resection were selected. After further removingredundant or poor-quality samples, 18,960 samples across40 tissue types were processed for data analysis.

Microarray data processing and analysisTo estimate expression level of each gene, microarray raw

CEL files were processed using the Michigan custom CDF(HGU133Plus2_Hs_ENTREZG, version 14; ref. 15) with anextended reference-based RMA summarization method(16). Fourteen-hundred samples from the entire data setwere randomly selected to estimate parameters of the RMAfit, which were then applied directly to the remainingmicroarray samples to produce gene-level expressions. Afterdata processing, expression levels of PIM1, PIM2, and PIM3were extracted for the samples described in Fig. 1 andSupplementary Fig. S1 and then visualized by boxplots inR (http://www.R-project.org).

Biochemical assays for PIM kinases and determinationof LGB321 inhibition constants

PIM1 (Invitrogen; PV3503), PIM2 (Invitrogen; PV3649),and PIM3 (Novartis) enzyme reactions were run in 50mmol/L Hepes, pH 7.5, 5 mmol/L MgCl2, 0.05% bovineserum albumin (BSA), 1 mmol/L dithiothreitol buffer witha biotinylated BAD peptide (b-RSRHSSYPAGT_NH2) andATP substrates. Reaction products were measured using thePerkinElmer AlphaScreen IgG Detection Kit (protein A)with anti-phospho-(Ser/Thr) AKT substrate antibody fromCell Signaling Technology. Apparent ATP Km calculationswere performed with Prism 5 (GraphPad Software Inc.)using the Michaelis–Menten equation. Ki measurementswere performed at high ATP concentrations. For PIM1, theATP concentration was 2,800 mmol/L, for PIM2 it was 500mmol/L, and for PIM3 it was 2,500 mmol/L. The apparent Ki

from these measurements was calculated using the Morri-son equation and converted to a true Ki using the Cheng–Prusoff relationship for an ATP site–competitive mecha-nism. Active site enzyme titrations were performed on allthree enzyme isoforms for use in the Morrison equation.

Morrison equation :

Vi=Vo ¼ 1� ½ðEþ Iþ Ki appÞ � sqrtðsqrðEþ Iþ KiappÞÞ�ð4� E� IÞ�=ð2� EÞ

Cheng--Prusoff equation : Kiapp ¼ Ki � ð1þ S=KmÞ

Translational RelevanceHere, we describe detailed biochemical, cellular, and

pharmacologic properties of a highly potent and selec-tive inhibitor (LGB321) of the three PIM kinases. Whentested in more than 500 cancer cell lines of diverseorigins, we demonstrated almost exclusive antiprolifera-tive activity in cells fromhematologic lineages, includingmultiple myeloma, acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), and non-Hodgkin lymphoma (NHL). This finding correlatedwithhigher levels of PIM kinases expression in these cancers,as compared with solid tumor cancers. To our knowl-edge, LGB321 is the first Pan-PIM inhibitor with activityon PIM2-dependent multiple myeloma cells. LGB321was effective in inhibiting tumor growth in an AMLxenograft model, in addition to a previously reportedmultiple myeloma model. Our findings suggest thatsingle-agent activity for Pan-PIM inhibitors is likely tobe observed in hematologic malignancies. On the basisof these findings, we initiated early clinical testing of ourdevelopment candidate in multiple myeloma, empha-sizing the translational relevance of the present work.

Garcia et al.

Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer ResearchOF2




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Figure 1. mRNA expression levels in cancer tissues. PIM1, PIM2, and PIM3 expression in patient samples from themajor hematologic cancers subtypes [ALL,N¼ 350; AML,N¼ 2,049; DLBCL-NHL,N¼ 640; andmultiple myeloma (MM),N¼ 982] as comparedwith the expression in normal bonemarrow (N¼ 81). Forcomparison 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 expressionof 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 whiskersextending from each box indicate expression values that are within 1.5 times the interquartile range (IQR) from the upper or lower quartile. Outliers thatare at a distance of greater than 1.5 � IQR from the box are plotted individually as plus signs.

Pan-PIM Kinase Inhibition in Hematologic Cancers

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Biochemical kinase specificity profileThe kinase specificity profile for LGB321 was determined

as previously described (17). Briefly, protein kinase activitywas assayed using either the LanthaScreen (www.invitro-gen.com) or the Caliper (www.caliperls.com) technologies.The biochemical activity for lipid kinases PI3Ka (phosphoi-nositide 3-kinase a), PI3Kb, Vps34, and PI4Kb was deter-mined by a luminescence assay based on ATP consumption(KinaseGlo; www.promega.com) with phosphoinositol asthe substrate, whereas PI3Kg , PI3Kdwere determined by theAdapta TR-FRET technology (www.invitrogen.com). Fordetermination of the biochemical activity of recombinantmTOR an antibody-dependent TR-FRET assay was usedwith 4EBP1 as substrate as well as an "ATP-binding assay,"which measures the occupancy of compounds in the ATPsite of mTOR.

Cell lines and reagentsTheKMS-11.luc humanmultiplemyeloma tumor cell line,

a KMS-11 clone expressing firefly luciferase, was obtainedfrom the University Health Network (UHN; Toronto,Ontario, Canada). KMS-11.luc and OPM-2 (DSMZ, Ger-many) were cultured in RPMI-1640 (American Type CultureCollection, ATCC), supplemented with 10% FBS. AML celllines KG-1 (ATCC) and MOLM-16 (DSMZ, Germany) werecultured in Iscove’s modified Dulbecco’s medium (IMDM;ATCC) or RPMI-1640 (ATCC), respectively, supplementedwith 20% FBS. The AML cell line P31/FUJ (Health SciencesResearch Resources Bank, Japan) was cultured in RPMI-1640(ATCC) media supplemented with 10% FBS. All cell culturemedia for in vitro work was additionally supplemented with20mmol/L glutamine, 1,000 IU/mLpenicillin and 1,000 mg/mL streptomycin. The origin and in vitromethods for the 947independent cancer cell lines in Cancer Cell Line Encyclope-dia (CCLE)hasbeenpreviously reported (18).Before implan-tation in vivo, KMS-11.luc and KG-1 cells were cultured inDulbecco’sModified EagleMedium (Corning) plus 10% FBSand 1% L-glutamine (Corning).

Cellular proliferation assaysTo assess the effect of LGB321 on proliferation, cells were

seeded in 96-well tissue culture plates followed by additionof compound that had been serially diluted to achieve afinal concentration range of 10 mmol/L to 2 nmol/L in 0.1%dimethyl sulfoxide (DMSO). After addition of LGB321 tocells, assay plates were returned to a humidified CO2 incu-bator (37�C; 5%CO2) for 3 days. To determine KMS-11.luccell growth, 100 mL per well of reconstituted CellTiter-Gloreagent was added to the cell assay plates. Assay plates werethen sealed and shaken on a DELFIA (PerkinElmer) plate-shaker for 10 minutes at 400 to 600 rpm. Plates were thenread on either a Microbeta Trilux (PerkinElmer) or Spectro-Max L (Molecular Devices) luminometer. Cell growth wasdetermined by comparing assay signals of LGB321 treatedcells with the control conditions of untreated cells (defining0% growth inhibition) or cells treated with KI-1, a potentnonspecific cytotoxic kinase inhibitor (defining 100%growth inhibition).

The activity of LGB321was also tested in the CCLE screen(18) and further testing was performed on an expandedpanel of hematologic cell lines. Cell lines were obtainedfrom commercial sources (ATCC or DSMZ) and were cul-tured in RPMI or IMDM plus 10% to 20% FBS (Invitrogen)as supplier recommended. All cell lines were thawed fromfrozen stock, grown at 37�C, 5% CO2, 95% relative humid-ity and cultured in T75 flasks using standard culture tech-niques. They were expanded for at least two passages beforebeing added in assay microtiter plates. Cell count wasmeasured using a CASY Model TT counter (Roche AppliedScience). All cell lineswere tested for and shown to be free ofmycoplasma using PCR detection. In addition, cell lineidentity was verified by single-nucleotide polymorphismgenotyping. Cell lines were dispensed into 384-well plates(Greiner Bio-One; #781098) with a final volume of 25 mLand concentrations ranging from 250 to 4,000 cells per wellin duplicate plates. Cell viability was assessed 3 hours afterseeding in the first plate (start value) and 120 hours afterseeding in the second plate by adding 25 mL Cell Titer-Glo(Promega; #G7571) per well. The doubling time was cal-culated for each condition and the optimal seeding densityleading to the shortest doubling time was selected forfurther profiling. Three hours after seeding, compoundswere transferred to the cells by delivering 5 mL per well ofthe intermediate dilution using the Velocity-Bravo Auto-mated Liquid Handling Platform (Agilent). This resulted ina final compound concentration range of 10 to 0.01 mmol/Lin a final volume of 25 mL and a final DMSO concentrationof 1%. The cell–compound mixture was incubated for 120hours. Cell Titer-Glo was added and luminescence was readon a Magellan plate reader (TECAN). On all plates, wellscontaining vehicle only were included. Cell lines wereseeded in parallel into a 384-well plate (Corning; #732-5528) in the same conditions and Cell Titer-Glo was added3 hours after seeding to evaluate cell viability before treat-ment (start value). Start values were subtracted and rawvalues were percentage normalized on a plate-by-plate basissuch that the median of the neutral control wells (i.e.,DMSO) is 100% growth and the D0 wells (optical densitymeasured at seeding time) is the 0% growth (100% inhi-bition). The crossing point defines the concentration atwhich growth is inhibited by 50% and is reported as GI50in this report.

Phosphoprotein assaysCommercial electrochemiluminescence (ECL) assay kits

fromMeso ScaleDiscovery (MSD)were used to quantify theeffects of LGB321 on the levels of phosphorylated S6RP andBAD in KMS-11.luc cells.

For in vitro assays, KMS-11.luc cellswere seeded in96-welltissue culture plates, and then LGB321 was serially dilutedand added to cell plates to achieve a final concentrationrange of 10 mmol/L to 2 nmol/L in 0.1% DMSO andincubated with cells for 1 hour at 37�C. Cells were pelletedby centrifugation for 7 minutes at 1,500 rpm, media wasgently aspirated, and MSD lysis buffer added. Cells werelysed by placing plates on a DELFIA (PerkinElmer) plate

Garcia et al.





shaker at 4�C and shaking the plates for 30 minutes at 600rpm.For in vivo assays, MDS lysis buffer (MSD) was added to

frozen pulverized tumor samples on ice and homogenateswere prepared using the MagNA Lyser bead instrument(Roche Applied Science) by disrupting the samples withfour cycles of 6,000 rpm for 30 seconds at 4�C. Supernatantswere created following centrifugation at 11,000 rpm for 15minutes at 4�C, and protein concentration determinedusing the BCA Protein Assay Kit according to the manufac-turer’s instructions (Pierce Chemical Company). For bothassays, samples were transferred to ECL assay plates previ-ously blocked with 3% BSA, sealed, and incubated at 4�Covernight while undergoing gentle shaking on a DELFIA(PerkinElmer) plate shaker. After overnight incubation,assay plates were processed according to themanufacturer’sinstructions.

LGB321 in vivo studiesAll studies were done in an Association for Assessment

and Accreditation of Laboratory Animal Care-accreditedanimal facility and in compliance with the ILAR Guide forthe Care and Use of Laboratory Animals. Scid/bg femalemice (10–12-week-old mice weighing about 20 g each;Charles River Laboratories) were housed up to 5 animalsper cage in clear polycarbonate microisolator cages with a12-hour light, 12-hour dark cycle at temperaturesbetween 70�F to 80�F, and 30% to 70% relative humidity.Food (Purina rodent chow pellets) and water were pro-vided ad libitum.For subcutaneous tumor models, cells were harvested at

80% to 90% confluency, washed, and resuspended in coldDulbecco’s Phosphate-Buffered Saline (without Ca2þ orMg2þ; CellGro) at a concentration of 5 � 107 cells/mL,mixed with an equal volume of Matrigel (Becton-Dickin-son) and then 0.2 mL (5 � 106 cells) was implantedsubcutaneously into the right flank of female Scid/bg mice.Tumor volume was measured in two dimensions using

digital calipers and calculated as ðlength�width2Þ � p=6.For pharmacodynamic and pharmacokinetic studies,

animals were enrolled on study when mean tumor vol-ume reached 250 to 400 mm3. For efficacy studies, ani-mals were randomized into groups when tumor volumereached 200 to 250 mm3. Tumor volume and bodyweights were captured and stored by StudyDirector soft-ware (StudyLog).LGB321 was formulated for oral administration in 50

mmol/L acetate buffer, pH4. The concentration of LGB321in plasma was determined following extraction in acetoni-trile using liquid chromatography and tandem mass spec-troscopy. Cytarabine (Hospira)was diluted in bacteriostaticwater and prepared fresh daily.

Statistical analysisFor in vivo studies, statistical significance of differences in

tumor volume was determined by a one-way ANOVA, andpair-wise comparisons weremade by the uncorrected FisherLSD posttest (GraphPad Prism software).

ResultsComprehensive analysis of PIM kinases mRNAExpression

Elevated expression of PIM kinases and their role incancer progression have been extensively reported in theliterature (4).However, a comprehensive evaluationof theirexpression in a large number of samples across normal andcancer tissue types has not been reported. Given that PIMsare constitutively active kinases (3), we reasoned that highmRNAexpression couldbeused to identify cancers inwhichPIM kinases are active. We evaluated datasets derived fromAffymetrix human genomeU133 arrays from public depos-itories for PIM kinases expression. All primary data werenormalized to allow direct comparison of expression acrossall tissues (Materials and Methods). Expression of PIM1,PIM2, and PIM3 was examined in normal and malignantsamples derived from 17 different tissue types (Supplemen-tary Fig. S1). Consistent with their known roles in cytokinesignaling in hematopoiesis (2), all three PIMs are expressedat higher levels in hematologic samples as compared withother tissues (Fig. 1 and Supplementary. Fig. S1). Increasedexpression of PIMs in most cancer types over their normaltissues counterpart is rather modest on average, suggestingthat PIM kinases expression is primarily hematologic line-age-specific.

Interestingly, across hematologic malignancies variousPIM isoforms are expressed at higher levels in acute lym-phoblastic leukemia (ALL), AML, multiple myeloma, orDLBCL-NHL samples, relative to samples of liver, lung,pancreas, prostate, and stomach cancers (Fig. 1). PIM2expression in multiple myeloma seems to be significantlyhigher than all other tissues examined, including normalbone marrow, whereas PIM1 expression is higher in AML,ALL, and DLBCL than in multiple myeloma (Fig. 1). Inaddition to the functional similarities as oncogenes (7, 8),this observation suggested that in order for a small-mole-cule inhibitor to have broad clinical utility the effectiveinhibition of all three PIM kinases (Pan-PIM) would berequired. Because we had observed that the multiple mye-loma KMS-11.luc cells are dependent on PIM2 for prolif-eration (14) and because PIM2 inhibition is the mostchallenging in the cellular context (as described in the nextsection), we chose this cell line to drive the development ofpotent Pan-PIM inhibitors in cells.

Biochemical activity and selectivity of the Pan-PIMinhibitor LGB321

We reasoned that an ATP-competitive inhibitor wasmostlikely to inhibit all three PIM kinases, because the ATP-binding pocket is highly conserved among the three PIMkinases and has some unique features relative to theATP pocket for other kinases (3). The development of acell-active ATP-competitive inhibitor for PIM2 was partic-ularly challenging, given its low ATP Km (4 mmol/L; Fig. 2B)as compared with the Km for PIM1 or PIM3 (400 and 40mmol/L, respectively; Fig. 2B). Thus, the desired inhibitorneeded to be potent enough to compete with the large






excess of cellular ATP (in the range of 1–10mmol/L; ref. 19),about 250- to 2,500-fold higher than the Km for ATP ofPIM2. We developed a series of highly potent and selectiveinhibitors of all three PIMkinases (20) and selected LGB321(Fig. 2A) as a tool compound with such characteristics. Theinhibition constant (Ki) for LGB321 was determined to bein the single-digit picomolar (pmol/L) range for each of thethree PIM kinases (Fig. 2B). Several groups have developedinhibitors to the PIMkinases in both academic and industrysettings (10–13); however, to our knowledge LGB321 is themost potent compound so far described. When directlycompared with two other PIM inhibitors tested in clinicaltrials, it is clearly more potent (Supplementary Fig. S2;ref. 20).

The selectivity of LGB321 was first determined in bio-chemical assays of a panel of seven lipid kinases and 68diverse protein kinases that included PIM2 (Table 1). In thispanel, only PIM2 was significantly inhibited by LGB321with an IC50 of <0.003 mmol/L, the lowest sensitivity rangefor the assay. Although biochemical potency in the range of4 to 10 mmol/L was demonstrated against eight otherkinases in this assay, these IC50 represent a greater than106-fold differential relative to the Ki on all three PIMkinases. The biochemical IC50 for all other kinases testedin this panel was >10 mmol/L (Table 1). We further evalu-ated the selectivity of LGB321 using the KINOMESCAN-binding displacement assay (Supplementary. Fig. S3;ref. 20). The results demonstrate that LGB321 has a highselectivity score with activity against only two other kinases(EGFR and ERK8) in this assay, in addition to the PIMkinases (Supplementary Fig. S3; ref. 20).

We further evaluated the activity of LGB321 at the cellularlevel on two of the potential off-targets. First, wemonitoredthe activity of LGB321 onGSK3b, themost potent off-targetkinase identified in the biochemical assay (Table 1). As seenin Supplemental Fig. S4 early compounds of the LGB321series showed cellular inhibition of GSK3b, as demonstrat-ed by the stabilization of b-catenin. However, compoundoptimization increased the biochemical selectivity andresulted in complete lack of activity of LGB321 on GSK3bin cells (Supplementary. Fig. 4). We next evaluated theactivity of LGB321 on EGFR, as it was identified as the mostpotent off-target in the KINOMESCAN-binding displace-ment assay. Despite this result, we found no evidence ofinhibitory activity in EGF signaling in the cellular context(Supplementary. Fig. S5). Together these results reinforcethat LGB321 is a highly potent and selective Pan-PIMinhibitor.

Cellular activity of LGB321The cellular activity of LGB321 was evaluated in KMS-11.

luc cells, a KMS-11 clone expressing firefly luciferase that wehave demonstrated previously to be dependent on PIM2kinase activity (14). Western blotting (Fig. 3A) was used todemonstrate that LGB321 inhibited the phosphorylation ofBAD at Ser-112 (a direct PIM kinase substrate; refs. 21–23)and two proteins downstream of the mTOR-C1 complex(S6K at Thr-389 and its substrate S6RP at Ser-235/6; ref. 24)in a concentration-dependent manner. The effect ofLGB321 in phosphorylation of S6K or S6RP is not due toan off-target effect on mTOR, because the compound wasinactive in the mTOR biochemical assay shown in Table 1and did not inhibit the phosphorylation of S6K or S6RP in293A/TSC2-null cells with constitutively activemTOR (14).

N

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Biochemical potency of LGB321 in PIM kinasesB

ATP Km LGB321 Ki

PIM1 400 µmol/L

4 µmol/L

40 µmol/L

1.0 pmol/L

PIM2 2.1 pmol/L

PIM3 0.8 pmol/L

Figure 2. LGB321 structure and biochemical properties. A, chemicalstructure of LGB321. B, summary of the ATP Km and LGB321inhibition constant (Ki) determined as described in Materials andMethods.

Table 1. LGB321Biochemical specificity profile

Kinase IC50 (mmol/L)

PIM2 <0.003GSK3b 4.4PKN1 4.4PKA 6.7S6K 6.8PKCa 7.0PKN2 7.2PKCt 7.7ROCK2 9.4

NOTE: Kinases with IC50 > 10 mmol/L: cABL(T315I), cABL,ALK, AURORA-A, BTK, CDK1B, CDK2A, CDK4D1, CK1,COT1, CSK, CaMK2, ERK2, EPHA4, EPHB4, FAK, FGFR1,FGFR2, FGFR3, FGFR4, FGFR3(K650E), FLT3, FYN, HCK,HER1, HER2, HER4, IGF1R, INS1R, IRAK4, JAK1,JAK2,JAK3, JNK2, JNK3, KDR, cKIT, LCK, LYN, cMET, MK2,MK5, MNK1, MNK2, PAK2, PDGFRa, PDK1, PI3Ka, PI3Kb,PI3Kd, PI3Kg , PI4Kb, PKBa, PLK1, RET, RON, cSRC, SYK,mTOR, TYK2, VPS34, WNK1, YES, ZAP70, p38a, p38g .

Garcia et al.





Thus, PIM kinases likely act upstream of mTOR to regulateits activity, a finding that agrees with our recent data dem-onstrating that PIM2 regulates mTOR through phosphor-ylation of TSC2 in the tuberous sclerosis complex (14).To better understand the relationship between the bio-

chemical and cellular potency of LGB321, we used quan-titative Meso Scale assays designed to measure levels ofphospho-BAD (pBAD) and phospho-S6RP (pS6RP). Sim-ilar to theWestern blot analysis results, these assays confirmthat LGB321 inhibits phosphorylation of both BAD andS6RP (at both the Ser-235/6 and Ser-240/6 sites, respec-tively) in a concentration-dependentmanner inKMS-11.luccells (Fig. 3B). In parallel experiments, we also determinedthe effect of LGB321on theproliferationofKMS-11.luc cellsusing CellTiter-GLO assays. The potency of LGB321 inmodulating PIM signaling correlated well with the inhib-itory effects on KMS-11.luc cell proliferation (Fig. 3B andC). We obtained similar results in KMS-26, KMS-34, andH929multiple myeloma cells, in which LGB321 inhibitionof proliferation and PIM signaling was observed (14).Interestingly, the inhibition of pBAD by LGB321 in thesemultiple myeloma cell lines did not correlate with signif-icant induction of PARP cleavage (14). Taken together, ourresults demonstrate that LGB321 is a potent and selectivePan-PIM inhibitor capable of inhibiting PIM kinase signal-ing and proliferation in PIM2-dependent multiple myelo-ma cell lines.

Activity of LGB321 in a large panel of cancer cell linesHaving established the potency, selectivity, and cellular

activity of LGB321, we proceeded to use it to test thehypothesis that PIM inhibition would have the greatestimpact on cell lines derived from hematologic lineages inwhich PIMkinases aremost highly expressed. To do this, wetook advantage of our high-throughput compound screenofmore than 500 cell lines from the CCLE (18). It should benoted that in addition to test compounds of interest, manyother well-characterized inhibitors and cancer drugs havebeen included in theCCLE screens, including panobinostat,erlotinib, and PLX4720 (18). These inhibitors serve ascontrols for specificity by showing differential activity incell lines of diverse cancer lineages or genetic backgrounds(18). LGB321 has been tested in this screen on threedifferent occasions, and in each case with similar results(data not shown). For simplicity, the data from a singlescreen are presented here to demonstrate the pattern ofLGB321 activity. As is apparent in Fig. 4A, LGB321 displaysvery limited activity in most cell lines derived from solidtumors, including breast, central nervous system, kidney,large intestine, liver, lung, ovarian, pancreas, prostate, andstomach cancer cell lines (Fig. 4A). The limited activityobserved in some lung cancer cell lines was not due topotential activity against EGFR because LGB321 does notinhibit EGFR signaling in cells (Supplementary Fig. S5).This was further supported by the fact that the lung cell linestested showed differential sensitivity to LGB321 and erlo-tinib, a potent and specific EGFR inhibitor (Supplemen-tary Table S1). In general, the lack of activity of LGB321 incell lines derived from solid tumors, including cancerindications for which evidence of roles for PIM kinaseshave been reported, was somewhat surprising. A possibleexplanation for this observation is suggested by the recentreport that PIM kinase inhibition may result in increasedexpression of several tyrosine kinase receptors in prostatecancer cells (25), which activates the mitogen-activatedprotein kinase and PI3K/AKT pathway as the main driversof proliferation in these cells. Nevertheless, our resultswith a highly potent and selective inhibitor suggest that insolid tumors PIM kinases are not the primary driver ofproliferation.

To further explore the LGB321 activity in hematologicmalignancies, we tested it in a screen that was adapted toevaluate the sensitivity of compounds on an extended panelof hematologic cell lines (Fig. 4B; Supplementary. TableS2). In this screen, cell lines from ALL, AML, multiplemyeloma, and B-cell NHLwere represented withmore than18 cell lines each. Multiple myeloma cells seem to be themost broadly sensitive to LGB321with 14of the 18 cell linestested having GI50 below 1 mmol/L (Fig. 4B; SupplementaryTable S2). Highly sensitive to LGB321was also identified inALL, AML, CML, and NHL cell lines. However, the responsewas less broad than in the multiple myeloma panel, with 4of 21 ALL, 15 of 26 AML, and 10 of 27 B-cell NHL cell linesshowing sensitivity to LGB321withGI50 below1mmol/L. Inaddition, activity was also observed inHodgkin lymphoma,CML, and T-cell NHL cell lines.However, the number of cell

A B

Concentration LGB321 (µmol/L)

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pons

e

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Actin

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CSubstrate EC50 (µmol/L) N

pBAD (S112) 0.010 ± 0.006 16

pS6RP (S235/6) 0.026 ± 0.013 4

pS6RP (S240/4) 0.038 ± 0.027 17

Proliferation 0.017 ± 0.011 26

100

80

pS6RP(S235/6) InhibitionpS6RP(S240/4) InhibitionpBAD(S112) InhibitionProliferation

60

40

20

0.001 0.01 0.1 1 100

Figure 3. Effect of LGB321 on PIM2 target modulation and proliferationin KMS-11.luc cells. A, Western blot analysis of KMS-11.luc cellstreated with a dose–response of LGB321 for 3 hours. B, quantitativeanalysis of the effects of LGB321 on KMS-11.luc proliferation andphosphorylation of S6RP andBAD; cells treatedwith a dose–response ofLGB321 were tested in 72-hour proliferation assays as well asquantitative ECL assays as described in Materials and Methods.C, summary of the quantitative cellular activities on LGB321 inKMS-11.luc cells.






lines representing these diseases is rather low. Furthermore,the genetic alterations frequently observed in these diseasesare represented in only a few cell lines. Thus, furtherexploration will be required to understand the role of PIMkinases in these malignancies and their diverse geneticbackgrounds. The broad distribution of LGB321 sensitivityamong hematologic cell lines, including both lymphoidand myeloid lineages, suggests that PIM kinase inhibitionmay have a broad application on the treatment of thesemalignancies.

Activity of LGB321 in vivoWe have reported elsewhere the antitumor activity of

LGB321 in the multiple myeloma KMS-11.luc xenograftmodel, together with a detailed characterization of itsmechanism of action inmultiple myeloma cells (14). Here,we focus on the characterization of the LGB321 activity inan AML xenograft model. Among the sensitive AML celllines tested in the expanded hematologic cell line panel(Fig. 4B; Supplementary Table S2), KG-1 cells were chosenas a model that readily grows in vivo. We first verified that

ALL

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mo

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Large intestine

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(Not-specified)

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0.5

-0.5

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-2.5

0

A

B

Cell lines

Figure 4. LGB321 screen in CCLE.A, cell lines are grouped and colorcoded according to theprimary siteof the tumor from whichthey were derived. To betterdemonstrate the differences insensitivity across the cell lines, thedirectionality of bars in the graph isbased on a concentration of 1mmol/L (log 0), with cell lines inwhich 50% growth inhibition wasachieved at lower concentrationsthan 1 mmol/L dropping down,and those in which it was higherthan 1 mmol/L rising up.Hematopoietic cancers are themost sensitive to PIM inhibition.B, LGB321 screen in an expandedpanel of cell lines from hematologicmalignancies. LGB321 is mostbroadly effective in MM. In AML, asubset of cell lines are verysensitive to LGB321. Sensitivity inALL, CML, Hodgkin lymphoma,and NHL cell lines was alsoobserved.

Garcia et al.





KG-1 cells were indeed sensitive to LGB321 in severalindependent CellTiter-GLO proliferation assays (GI50 of0.08 � 0.07 mmol/L; N ¼ 7). We then tested whether PIMinhibition in KG-1 cells resulted in modulation of pBADand the mTOR signaling pathway and found that indeedLGB321 effectively inhibited both signaling pathways

(Fig. 5A). Having established in vitro the responses ofKG-1 cells to PIM inhibition, we proceeded to evaluatedin vivo the LGB321 pharmacokinetic–pharmacodynamicrelationship in KG-1 subcutaneous tumors (Fig. 5B).Following a single oral dose of LGB321 (30 or 100mg/kg), at the indicated time points plasma and tumor

Figure 5. LGB321 activity in AMLxenografts and demonstration of invivo synergy with cytarabine. A,modulation in vitro of pBAD andmTOR signaling in KG-1 cells. B,single-dose pharmacodynamic/pharmacokinetic of LGB321 inmicebearing subcutaneous KG-1tumors. C, LGB321 efficacy assingle agent and in combinationwith cytarabine. LGB321wasdosedby oral gavage with vehicle,LGB321 at 30, 100 mg/kg oncedaily (QD), cytarabine at 100 mg/kgQD by intraperitoneal injection, orwith a combination of cytarabineand 30 or 100 mg/kg LGB321(n ¼ 9/group). Although 30 mg/kgQD of LGB321 resulted insignificant tumor growth inhibition(�, P < 0.05), 100 mg/kg QDachieved thegreater effect of partialregression. In contrast, cytarabinehas no effect in this model as asingle agent. However, whencombined with 30 mg/kg QD ofLGB321, a synergistic effect wasachieved resulting in tumorregression (P < 0.05). Whencytarabine was combined with 100mg/kg LGB321, this also resulted inincreased tumor regression,though this result was notstatistically significant comparedwith the 100 mg/kg LGB321 groupalone. The combination of LGB321and cytarabine led to significantbody weight loss after 5 successivedays of dose, and the combinationgroups were granted a 2-daydosing holiday beforeadministration continued.






samples were collected for pharmacokinetics and phar-macodynamics analysis, respectively. PIM kinase inhibi-tion was determined by assessing the modulation ofpS6RP and pBAD in tumor lysate using the quantitativeMeso Scale assay and the results were expressed as a ratioof their phosphorylated to unphosphorylated forms (Fig.5B). A dose-dependent increase in the plasma concentra-tion of LGB321 was observed (Fig. 5B). At the 100 mg/kgdose, the plasma concentration was maintained at higherlevels through 24 hours, whereas at the 30 mg/kg dose agradual reduction in the plasma concentration occurred.The modulation of pS6RP in tumors was also dose-dependent with more sustained inhibition at 100 mg/kgbut only transient inhibition at 30 mg/kg. In contrast,both doses achieved equally significant and sustainedpBad inhibition through 24 hours. This observationraises the question of which target modulation markerwould better predict the in vivo efficacy of LGB321.

To address this question, we tested the same 30 and 100mg/kg doses of LGB321 in a daily regimen of oral dosing inan 11-day efficacy study. At the 30 mg/kg daily regimen ofLGB321, we observed near stasis with only minor increasein tumor volume relative to the vehicle control (Fig. 5B). Incontrast, slight tumor regression was observed with the 100mg/kg daily regimen. These data establish that the extentand duration of pS6RP, and not pBad, modulation corre-lateswith efficacy in this AMLmodel, a finding in agreementwith our previous data in multiple myeloma models (14).We have also recently reported similar in vivo activity ofLGB321 in the AML EOL1 xenograft model (20). Here, wetested the combination of LGB321 with the nucleosideanalog cytarabine, a standard-of-care in the clinical treat-ment of AML. KG-1 tumors did not exhibit a significantresponse to cytarabine alone when delivered at 100 mg/kgdaily, a dose that results in clinically relevant exposures(26). When 30 mg/kg LGB321 daily dose was combinedwith 100mg/kg daily dose of cytarabine, a synergistic effectresulting in slight regression was achieved (Fig. 5B). Whencombined with the higher dose of LGB321, the synergisticeffect was not statistically significant, given that regressionwas achieved with 100 mg/kg LGB321 alone. However, thecombination of cytarabine with LGB321 induced signifi-cant body weight loss following the fifth day of adminis-tration (not shown), which required dosing to be haltedonly in the combination arms for 2 days before resumingtreatment. These results demonstrate the efficacy of LGB321both as a single agent as well as in combination withcyatarabine, even in a model that is refractory to thisstandard of care as a single agent.

DiscussionHere, we demonstrated that each of the three PIM kinase

family members is most highly expressed in at least onesubtype of hematologic cancer. This observation coupledwith the fact that PIM family members can substitute foreach other to generate lymphomas (6, 7) indicates that toeffectively treat hematologic cancers, an inhibitor of allthree isoforms is required. Furthermore, we describe

LGB321, a potent and selective inhibitor of the PIM kinasefamily members capable of inhibiting PIM2 at the highcellular concentrations of ATP (20). Because LGB321 is apicomolar inhibitor of all three PIM family members, wecarefully evaluated its selectivity against other kinases inboth biochemical and cellular contexts. The selectivity wastested biochemically in a panel of 75 kinases (Table 1) andby binding displacement assays in a panel of 386 kinases(Supplementary Fig. S3; ref. 20). Furthermore, cellularassays for the closest off-target kinase in each of these panels(Supplementary Figs. S4 and S5) were used to demonstratethat LGB321was inactive on these kinases at concentrationswell above the on-target activities on PIM2-dependent cells(Fig. 3). Selectivity was further evident by the almost exclu-sive activity of LGB321 in hematologic cell lines amongmore than 500 cell lines in the CCLE.

LGB321 as single agent was well-tolerated in vivo aftermultiple doses, a finding predicted by the viability andfertility of mice with knockout of the three PIM kinasegenes (27). LGB321 can be administered orally to reachplasma exposures that allowed us to evaluate the relation-ship between pharmacokinetics, pharmacodynamics, andefficacy of PIM inhibition in xenograft models. Althoughoral administrationwith LGB321 leads to a dose-dependentincrease in plasma exposure, the increased exposure wasachieved through extended plasma drug concentrations,rather than successive increases in peak plasma concentra-tion, as exhibited by differences between the 30 and 100mg/kg doses (Fig. 5B). The pharmacokinetic properties ofLGB321 at higher doses are somewhat advantageous, as theextended exposure is concomitant with sustained targetinhibition as evidenced by pS6RP inhibition. The maximalin vivo effect was slight tumor regression at the 100mg/kg/dregimen in the AML KG-1 model (Fig. 5C) and tumor stasisin the KMS-11.luc (14) and the EOL-1 models (20). As thisdose is near themaximal tolerated dose, we could not assesswhether LGB321 would achieve tumor regression at higherexposures. Nevertheless, stasis or slight regression are con-sistent with our results that LGB321 leads to inhibition ofcell proliferation and not to apoptosis as assessed by PARPcleavage in the KMS-11.luc model (14). In the KG-1model,tumor regression of approximately 50%was observedwhenLGB321 was coadministered with cytarabine, a nucleosideanalog that is integral to the current standards of care inAML (28). Interestingly, in this model cytarabine alone hadno effect, highlighting the suggestion that the combinedused of this agent and PIM inhibitors could enhance theclinical response (29).

The higher level of PIMmRNA expression in hematologictissues is consistent with a distinct role for PIM kinases inhematologic cancers. We demonstrated that PIM kinaseinhibition has an antiproliferative effect primarily in hema-tologic cell lines, with very limited activity in cell lines fromsolid tumors. Although the role of PIM kinase in some solidtumors has been extensively described in the literature(2, 4), our data suggest that PIM kinases play a moresignificant role in promoting proliferation in hematologicmalignancies than in solid tumors. Interestingly, PIM

Garcia et al.





kinases were identified as oncogenes by insertional muta-genesis screens in lymphomas frommurine leukemia virus–infected mice (8, 30), but not in tumors induced by themouse mammary tumor virus (31). These observationssuggest that PIM kinases, in addition to being specificallyexpressed in the hematologic lineage, are hematologiclineage-specific oncogenes. Our results that the inhibitoryactivity of LGB321 is observed primarily in cell lines ofhematologic lineage further strengthen the notion that PIMkinases play a lineage-related role in promoting prolifera-tion of some hematologic cancers and suggest that poten-tially single-agent activity may be observed in these cancers.In aggregate, our results strongly suggest that the use of

potent and selective pan-PIM inhibitors, either as singleagent or in combination with other agents, will be veryuseful for the treatment of hematologic malignancies ingeneral, and inmultiplemyeloma andAML inparticular. Totest this hypothesis in humans, we have initiated the phase Iclinical testing of our development candidate LGH447 inrelapsed and/or refractory multiple myeloma.

Disclosure of Potential Conflicts of InterestP.D. Garcia has ownership interest (including patents) inNovartis Shares.

J.L. Langowski is an investigator in Novartis. J. Lan is a research investigatorIII in Novartis. M. Lindvall has ownership interest (including patents) inNovartis. J. Holash has ownership interest (including patents) in Novartisstock. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: P.D. Garcia, J.L. Langowski, C. Fanton, T. Zavor-otinskaya, X.-H. Niu, J. Lan, M. Lindvall, J. Holash, M.T. BurgerDevelopment of methodology: P.D. Garcia, J.L. Langowski, M. Chen, J.Castillo, M. Ison, Y. Dai, X.-H. Niu, S. Basham, J. Chan, J. Yu, M. Doyle, P.Feucht, C. Fritsch, E. Pfister, J.A. AycinenaAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): J.L. Langowski, Y. Wang, C. Fanton, T. Zavor-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. ZanglAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): P.D. Garcia, J.L. Langowski, M. Chen, C.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.Zang, J. HolashWriting, review, and/or revision of the manuscript: P.D. Garcia, J.L.Langowski, C. Fanton, X.-H. Niu, S. Basham, M. Doyle, P. Feucht, J. HolashAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Castillo, M. Ison, Y. Dai, X.-H.Niu, J. Narberes, T. Tsang, W. Han, G. NishiguchiStudy supervision: P.D. Garcia, T. Zavorotinskaya, X.-H. Niu, S. Basham

AcknowledgmentsThe authors thank Dr. Suzanne Trudel at the UHN for generously

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 alsothank Gary Vanasse, Chuck Voliva, Emma Lees, andWilliam Sellers for theirsupport and critical reading of the article.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 29, 2013; revised December 6, 2013; accepted January 7,2014; published OnlineFirst January 28, 2014.

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Garcia et al.





Published OnlineFirst January 28, 2014.Clin Cancer Res Pablo D. Garcia, John L. Langowski, Yingyun Wang, et al. Treating Hematologic CancersPan-PIM Kinase Inhibition Provides a Novel Therapy for

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