The Rockefeller University Press $30.00
J. Exp. Med. Vol. 208 No. 9 1799-1807
Brief Definitive Report
Molecular signaling pathways are promising
targets in cancer therapy, but resistance often
thwarts clinical success. Acquired mutations of
drug targets, feedback activation of oncogenic
signals, and redundant signaling pathways are
important causes of resistance, and cocktails of
multiple inhibitors are considered one potential
solution (Sawyers, 2007). For example, the rapa
mycin analogues (rapalogs) are potent inhibi
tors of mTORC1 with promising antitumor
activity against some cancers (Dancey, 2010).
mTORC1 blockade by rapamycin interferes
with the activation of capdependent transla
tion and exploits a cancer cell’s dependence on
increased translation of certain oncoproteins
(Wendel et al., 2007; Sonenberg and Hinnebusch,
2009). In animal models, rapamycin dramatically
enhances the effectiveness of DNAdamaging
chemotherapy (Wendel et al., 2004). However,
in clinical trials in nonHodgkin’s lymphoma
(NHL), rapalogs have failed to show durable
clinical benefit for most patients (Dancey et al.,
2009; Hess et al., 2009; Smith et al., 2010). The
causes are illunderstood, and new insight should
enable better therapies.
Abbreviations used: ABC,
activated B cell; CLL/SLL,
chronic lymphocytic leukemia/
small lymphocytic lymphoma;
DLBCL, diffuse large B cell
lymphoma; FL, follicular
lymphoma; GC, germinal center;
HPC, hematopoietic progenitor
cell; MCL, mantle cell lym
phoma; NHL, nonHodgkin’s
lymphoma; OS, overall survival;
shRNA, small hairpin RNA;
TMA, tissue microarray; TTE,
time to event.
Targeting cap-dependent translation blocks
converging survival signals by AKT and PIM
kinases in lymphoma
Jonathan H. Schatz,1,4 Elisa Oricchio,1 Andrew L. Wolfe,1,7 Man Jiang,1
Irina Linkov,5 Jocelyn Maragulia,4 Weiji Shi,6 Zhigang Zhang,6
Vinagolu K. Rajasekhar,2 Nen C. Pagano,3 John A. Porco Jr.,8
Julie Teruya-Feldstein,5 Neal Rosen,3,4 Andrew D. Zelenetz,4
Jerry Pelletier,9,10 and Hans-Guido Wendel1
1Cancer Biology and Genetics Program, 2Stem Cell Center and Developmental Biology Program, and 3Program in Molecular
Pharmacology, Sloan-Kettering Institute for Cancer Research, New York, NY 10065
4Department of Medicine, 5Department of Pathology, and 6Department of Biostatistics and Epidemiology, Memorial Sloan-
Kettering Cancer Center, New York, NY 10065
7Weill Cornell Graduate School of Medical Science, New York, NY 10065
8Department of Chemistry, Center for Chemical Methodology and Library Development, Boston University, Boston, MA 02215
9Department of Biochemistry and 10Rosalind and Morris Goodman Cancer Center, McGill University, Montreal, Quebec
H3G 1Y6, Canada
New anticancer drugs that target oncogenic signaling molecules have greatly improved the
treatment of certain cancers. However, resistance to targeted therapeutics is a major
clinical problem and the redundancy of oncogenic signaling pathways provides back-up
mechanisms that allow cancer cells to escape. For example, the AKT and PIM kinases pro-
duce parallel oncogenic signals and share many molecular targets, including activators of
cap-dependent translation. Here, we show that PIM kinase expression can affect the clini-
cal outcome of lymphoma chemotherapy. We observe the same in animal lymphoma models.
Whereas chemoresistance caused by AKT is readily reversed with rapamycin, PIM-mediated
resistance is refractory to mTORC1 inhibition. However, both PIM- and AKT-expressing
lymphomas depend on cap-dependent translation, and genetic or pharmacological blockade
of the translation initiation complex is highly effective against these tumors. The therapeu-
tic effect of blocking cap-dependent translation is mediated, at least in part, by decreased
production of short-lived oncoproteins including c-MYC, Cyclin D1, MCL1, and the PIM1/2
kinases themselves. Hence, targeting the convergence of oncogenic survival signals on
translation initiation is an effective alternative to combinations of kinase inhibitors.
© 2011 Schatz et al. This article is distributed under the terms of an Attribution–
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The Journal of Experimental Medicine
Targeting survival signals in lymphoma | Schatz et al.
and Table S2). The same analyses of 116 DLBCL patients treated
between 1989 and 2008 showed differences that did not reach
statistical significance in OS (P = 0.1678) or TTE (P = 0.4461;
Fig. 1, H and I; and Table S3). Similarly, another group re
cently reported association of PIM2 with outcome in DLBCL
(GómezAbad et al., 2011). All but three of the DLBCL pa
tients were treated with upfront chemotherapy, including
doxorubicin in 88% of patients. Statistical analyses for each PIM
kinase analyzed as a single variable or coexpression of PIM1/2
in FL and DLBCL are available in Table S4 and Table S5.
PIM promotes the development of drug-resistant
lymphomas in vivo
To study the function of PIM kinase activity in lymphomas,
we modeled its effects in murine models of aggressive pre–B
cell (Adams et al., 1985) and indolent follicular lymphoma
(Egle et al., 2004). In brief, we used adoptive transfer of
Eµ-Myc or VavPBcl2 transgenic hematopoietic progenitor
cells (HPCs; Wendel et al., 2004) expressing AKT, Pim2, or
vector into lethally irradiated, syngeneic wildtype recipients
and monitored the animals for lymphomas (Fig. 2 A). PIM1
and PIM2 are highly homologous, therefore we did not ex
amine PIM1 separately (Nawijn et al., 2011). Both Pim2
(n = 12; P < 0.0001) and AKT (n = 30; P < 0.0001) accelerated
disease onset compared with controls (n = 64; P = 0.1209
Pim2 vs. AKT; Fig. 2 A; Verbeek et al., 1991; Wendel et al.,
2004). Immunoblotting confirmed expression of AKT and
Pim2 and translational activation by both kinases as indicated
by increased phosphorylation of 4EBP1 and ribosomal pro
tein S6 (Fig. 2 C). Histopathology and surface marker analysis
revealed that Pim2 and AKTexpressing tumors were indis
tinguishable from aggressive pre–B cell lymphomas (Fig. 2 B
and unpublished data). The VavPBcl2 model is a genetically
and pathologically accurate model of FL, and both Pim2
(P < 0.0001) and AKT (P = 0.0292) accelerated development
compared with vector of a slowly proliferating B cell lymphoma
with splenic involvement and increased peripheral lympho
cyte counts (unpublished data). Hence, Pim2 and AKT acti
vate protein translation and promote lymphomagenesis in
mouse models of aggressive and indolent lymphoma.
Next, we examined how PIM and AKT affect treatment
responses in vivo. In brief, we transplanted aggressive Eµ-Myc
lymphomas with defined genetic alterations into nonirradi
ated recipients, and then treated with 10 mg/kg doxorubicin
once lymphomas had developed (Wendel et al., 2004). A side
byside comparison of chemosensitive Eµ-Myc/Arf/ tumors
(control; n = 44) with Eµ-Myc/Pim2 (n = 6), or Eµ-Myc/AKT
(n = 30) lymphomas, revealed early relapse and shortened sur
vival with Pim2 and AKTexpressing tumors (Fig. 2 D; P =
0.0145 Pim2 vs. Arf, P<0.0001 AKT vs. Arf). Rapamycin
alone had little effect on any tumor (Fig. 2 E). However, combi
nations of rapamycin with doxorubicin caused dramatic re
sponses in AKT lymphomas, but had no effect on Pim2expressing
tumors (Pim2, n = 13; AKT, n = 21; control, n = 28; P < 0.0001,
Pim vs. AKT; Fig. 2 F). Hence, chemoresistance caused by AKT
but not by Pim2 is readily reversed by mTORC1 inhibition.
Multiple oncogenic signaling pathways cause aberrant
activation of protein translation in cancer cells, including RAS,
PI3K–AKT, MAPK, and the PIM kinases (Sonenberg and
Hinnebusch, 2009). The PIM kinases were identified in a ge
netic screen. They promote cell growth and survival and share
many targets, including regulators of protein translation, with
the better studied AKT/PKB kinases (Nawijn et al., 2011).
PIM kinases are induced by cytokine signals and, unlike AKT
do not require posttranslational modifications for activity
(Fox et al., 2003). Activation of capdependent translation via
derepression of the translation factor eIF4E is a critical output
of both AKT and PIM signaling in cancer (Wendel et al.,
2004; Hammerman et al., 2005). PIM1 and PIM2 are widely
expressed in cancer; PIM3 is restricted to certain solid tumors
(Nawijn et al., 2011). Accordingly, PIM inhibitors have been
developed, but clinical trials were terminated early because of
cardiac toxicity (Morwick, 2010). Our study explores the
clinical impact of PIM1/2 expression in NHL, and we demon
strate that inhibition of capdependent translation is an effec
tive therapy alternative to combinations of kinase inhibitors.
RESULTS AND DISCUSSION
PIM1 and PIM2 are widely expressed in NHL and affect
the outcome of follicular lymphoma (FL)
We found widespread expression of PIM1 and PIM2 across
multiple subtypes of NHL. Immunohistochemical staining of
tissue microarrays (TMA) reveals that PIM1 is expressed in
87% of mantle cell lymphomas (MCL; Hsi et al., 2008), 76%
of chronic lymphocytic leukemia/small lymphocytic lym
phoma (CLL/SLL; Chen et al., 2009), and 48 and 42% of dif
fuse large B cell lymphoma (DLBCL) and FL, respectively.
PIM2 is detected in 42% of DLBCL and between 24% and
30% of FL, MCL, and CLL/SLL (Fig. 1, A–E; and Table S1).
Similarly, PIM1/2 mRNA levels are highly expressed in the
activated B cell (ABC) type, rather than the germinal center
(GC) type of DLBCL (Alizadeh et al., 2000; Rosenwald et al.,
2003; Basso et al., 2005; Lenz et al., 2008; unpublished data).
PIM2 is abundantly expressed across a panel of human lym
phoma cell lines, whereas PIM1 is coexpressed in some, and
immunoblots on mouse pro–B cells and EµMyc lymphomas
confirm PIM1/2 induction by cytokine signals (Fox et al.,
2003; unpublished data).
PIM expression affects the outcome of therapy in follic
ular lymphoma patients. First, we analyzed pretreatment follic
ular lymphoma samples from 66 patients treated at Memorial
SloanKettering Cancer Center (MSKCC) between 1984
and 2000 (Table S2). All but five of these patients received
chemotherapy, including doxorubicin in 61% of patients. In
this cohort, time to event (TTE) and overall survival (OS)
were significantly better for patients whose tumors were
PIMnegative (PIM, no PIM1 or PIM2) compared with
patients whose tumors were PIMpositive (PIM+, PIM1,
PIM2, or both; P = 0.0113 for TTE, P = 0.0372 for OS for
PIM+ vs. PIM tumors). The mean age was 60.9 and 52.6 yr
for the groups, respectively; however, age alone did not ex
plain the difference in outcome (P = 0.13; Fig. 1, F and G;
JEM Vol. 208, No. 9
Brief Definitive Report
are rapidly enriched under rapamycin treatment (Fig. 3, A and
inset). Pim2 causes partially rapamycininsensitive increases
in the phosphorylation of 4EBP1, eIF4E, and Bad, whereas
S6 phosphorylation remains sensitive to rapamycin (Fig. 3 B).
The capbinding protein eIF4E is the ratelimiting factor in
capdependent translation that is activated by phosphoryla
tion of its inhibitor 4EBP1 and can be further enhanced by
direct eIF4E (S209) phosphorylation (Wendel et al., 2007;
Sonenberg and Hinnebusch, 2009). Profiles of ribosome load
ing on mRNAs (polysome profiles) indicate the efficiency of
protein translation. Polysome profiles on parental and Pim2
expressing EµMyc/Tsc2/ lymphoma cells reveal a partially
rapamycinrefractory increase of protein
translation in Pimexpressing lym
phomas (Fig. 3 C). Accordingly, both
Pim and direct expression of eIF4E
protect against rapamycin and have a
similar effect in cells treated with the
TOR kinase inhibitors PP242 and
Torin1 (Feldman et al., 2009; Thoreen
et al., 2009; Fig. 3 D). By comparison,
a small hairpin RNA (shRNA) against
BAD showed no protective effect dur
ing rapamycin treatment (unpublished
data). To examine whether PIM
expressing tumors remained depen
dent on capdependent translation, we
tested the antiproliferative effects of
a constitutively active inhibitor of
eIF4E (4E-BP1-4A) that acts down
stream from mTORC1 (Rong et al.,
2008). Surprisingly, parental Eµ-Myc/
Tsc2/ lymphomas and Pim2 ex
pressing Eµ-Myc/Tsc2/ cells were
equally sensitive to direct inhibition of
eIF4E and cells expressing 4EBP1/
GFP were rapidly depleted from
a mixed population, but had little
effect in nontransformed cells (Fig. 3 E
and unpublished data). Hence, PIM2
readily bypasses mTORC1 inhibition,
but is unable to protect lymphoma
cells from the effects of direct transla
PIM-expressing lymphomas remain dependent on eIF4E
and cap-dependent translation
We examined how PIM bypasses mTORC1 inhibition in
rapamycinsensitive Eµ-Myc/Tsc2/ lymphomas (Mills et al.,
2008). TSC2 is the Rheb GTPaseactivating protein and acts
as a negative regulator of mTORC1 activation by Rheb (Tee
et al., 2003; Mavrakis et al., 2008). Accordingly, tumors arising
in Tsc2 deficient animals show an mTORC1dependent and
rapamycinsensitive activation of capdependent translation.
Pim2 expression in Eµ-Myc/Tsc2/ cells abrogates rapamy
cin sensitivity, and in mixed populations of parental and Pim2/
GFPexpressing Eµ-Myc/Tsc2/ cells the Pim2/GFP cells
Figure 1. PIM kinase expression affects
the outcome of lymphoma therapy. (A and
B) DLBCL TMAs stained for PIM1 (A) and PIM2
(B). (C and D) Representative tumor cores for
each PIM histology score (0–2). (E) Pie graphs
showing breakdown of PIM1/2 TMA scores by
disease; see also Table S1. (F) TTE analysis
after primary therapy in follicular lymphoma
(n = 66). (G) OS analysis from date of diagno-
sis in follicular lymphoma. (H and I) TTE (H)
and OS (I) in DLBCL (n = 116).
Targeting survival signals in lymphoma | Schatz et al.
absence of translational activation (Wendel et al., 2004; Fig. 4 C).
Moreover, silvestrol is also far superior to two recently devel
oped PIM inhibitors in human lymphoma cells. In brief, we
tested SGI1776, the only PIM inhibitor that has entered
clinical trials (biochemical IC50 for PIM1, 15 nM; PIM2, 363
nM), and SGI1773 (biochemical IC50 for PIM1, 2 nM;
PIM2, 43 nM); both drugs were developed and supplied to us
by SuperGen Inc. (Morwick, 2010). The PIM kinase inhibi
tors induced cell death in various human lymphoma cells at
concentrations between 1–10 µM; in comparison, silvestrol
had the same cell kill at 1–10 nM (Fig. 4 D). In animals, sil
vestrol was able to reverse Pim2mediated rapamycin resistance
Silvestrol is a small molecule inhibitor of cap-
Silvestrol was identified in a screen for inhibitors of eIF4A,
the RNA helicase component of the translation initiation
complex that is thought to unwind an mRNA’s 5UTR
(Bordeleau et al., 2008). Consistent with our genetic data
using a constitutive 4E-BP1 construct, we found that Pim2 is
unable to protect Eµ-Myc/Tsc2/ cells from silvestrol alone
or in combination with rapamycin (Figs. 4, A andB). Silvestrol
kills parental and Pim2expressing Eµ-Myc/Tsc2/ cells at
nanomolar concentrations in vitro, but is inactive against 3T3
fibroblasts and Myc/Bcl2 lymphomas tumors that arise in the
Figure 2. Pim2 and AKT in a mouse lymphoma model. (A) Eµ-Myc HPCs expressing Pim2, AKT, or vector were transplanted into lethally irradiated syn-
geneic wild-type mice. Tumor onset in Pim2 (green; n = 12), AKT (red; n = 30) and vector (black; n = 64 recipients). (B) Histological and immunohistochemical
analyses of indicated Eµ-Myc lymphomas. Bar, 20 µm. (C) Immunoblot analyses of indicated Eµ-Myc lymphomas. (D–F) Time to relapse in animals bearing
Eµ-Myc/Arf/ (control, black line), Eµ-Myc/Pim2 (green), and Eµ-Myc/AKT (red) lymphomas treated with doxorubicin (D; control, n = 44; Pim2, n = 6; AKT,
n = 30) or rapamycin (E; control, n = 27; Pim2, n = 7; AKT, n = 18) or a combination of both drugs (F; control, n = 28; Pim2 ,n = 13; AKT, n = 21).
JEM Vol. 208, No. 9
Brief Definitive Report
oncoproteins including c-MYC, MCL1 and Cyclin D1
(Sonenberg and Hinnebusch, 2009). Treatment of PIM
expressing human lymphoma cells with the PIM inhibitor
SGI1773 (10 µM) somewhat reduced Cyclin D1, but had no
effect on c-MYC or MCL1 (Fig. 4 F). In contrast, silvestrol
(10 nM) caused almost complete loss of Cyclin D1, c-MYC,
and MCL1. Moreover, silvestrol completely ablated the ex
pression of both PIM1 and PIM2 kinases (Fig. 4 F). Silvestrol
had similar effects on PIM expression in DoHH2 and
SuDHL10 (Fig. 4 G). This is consistent with the known
short halflife of PIM1 and PIM2 and indicates that PIM ex
pression is controlled, at least in part, by capdependent transla
tion (Hoover et al., 1997). This dual effect of translation inhibition
on PIM and its downstream targets likely accounts for silves
trol’s dramatic activity against mouse and human lymphomas.
Our study provides new insight into oncogenic kinases in
human lymphoma. The constitutively active PIM1 and PIM2
kinases are abundantly expressed across several subtypes of
NHL, and in follicular lymphoma, PIM positivity identifies
patients at risk of early relapse and shortened
survival and who may require specific treat
ment. Similarly, in DLBCL, PIM1/2 expression
is associated with the prognostically unfavorable
ABC subtype (Alizadeh et al., 2000; Rosenwald
et al., 2003; Wright et al., 2003; Basso et al.,
2005; Poulsen et al., 2005; Lenz et al., 2008;
GómezAbad et al., 2011). Although clinical
data on the effect of PIM expression on rapalog
treatment are not yet available, our data and
other evidence indicate that neither rapalogs
nor the newer TORkinase inhibitors will be
and did not cause overt toxicity at an effective dose (0.2 mg/kg,
d17), consistent with published silvestrol toxicity studies,
showing no major adverse effects at this dose and duration of
treatment (Cencic et al., 2009). In brief, animals bearing
parental Tsc2deficient tumors cells (n = 9) remained relapse
free for up to 3 wk after rapamycin, whereas Eµ-Myc/Tsc2//
Pim2 lymphomas (n = 9) showed no response or relapsed
early (P = 0.0006; Fig. 4 E). The addition of silvestrol to rapa
mycin treatment restored rapamycin sensitivity, and Eµ-Myc/
Tsc2/Pim2 tumorbearing animals remained relapse free
for as long as sensitive controls (P = 0.7219; Fig. 4 E). Hence,
the translation inhibitor silvestrol has good activity active
against human lymphoma cells and can overcome PIM
mediated resistance in vivo.
Translation is required to maintain expression
of oncoproteins including c-MYC and PIM
In cancer the activation of capdependent protein trans
lation by AKT or PIM ensures the expression of shortlived
Figure 3. PIM confers resistance to mTOR inhibi-
tion, but not to genetic blockade of cap-dependent
translation. (A) Cell viability in vitro comparing rapamycin-
sensitive Eµ-Myc/Tsc2/ lymphomas expressing vector-
GFP, Pim1-GFP, or Pim2-GFP under rapamycin treatment.
(inset) Enrichment of the Pim2-GFP–expressing subpopu-
lation of Eµ-Myc/Tsc2/ cells upon rapamycin
exposure in vitro. (B) Immunoblot on lysates of
Eµ-Myc/Tsc2//vector or Eµ-Myc/Tsc2//Pim2 cells
treated with vehicle (U) or rapamycin (R), and probed for
the indicated proteins. (C) Polyribosome profiles gener-
ated from untreated and rapamycin-treated Eµ-Myc/
Tsc2/ and Eµ-Myc/Tsc2//Pim2 tumors, indicating the
ability of PIM2 to stimulate translation in a partially
rapamycin-resistant manner (absorbance at 254 nm).
(D) Enrichment of populations Eµ-Myc/Tsc2/ cells
expressing vector-GFP (black), Pim2-GFP (orange), and
eIF4E-GFP (blue) and treated with rapamycin or the
TOR-kinase inhibitors PP-242 and torin (mean fold
change and SEM of 5 separate experiments; * indicates
significance [P < 0.05] vs. vector). (E) Enrichment or loss
of subpopulations of Eµ-Myc/Tsc2/ and Eµ-Myc/Tsc2//
Pim2 cells engineered to express vector encoding GFP or
a constitutively active inhibitor of eIF4E (4E-BP1-4A-GFP)
during culture in vitro (mean results and SEM of three
Targeting survival signals in lymphoma | Schatz et al.
(Sonenberg and Hinnebusch, 2009). Both kinases can limit
the effectiveness of chemotherapy, and although the effects of
AKT are readily reversed by blocking mTORC1 and transla
tion with rapamycin (Wendel et al., 2004), PIMexpressing
tumors remain refractory and are able to maintain trans
lation in an mTORC1independent manner. However, PIM
expressing tumor cells continue to depend on translational
activation, and they are therefore sensitive to small molecules
that directly target the translation initiation complex down
stream from mTORC1. For example, silvestrol, an inhibitor
of the eIF4A RNA helicase (Bordeleau et al., 2008), is highly
active against PIMexpressing tumors (Fox et al., 2003;
Hammerman et al., 2005). PIM kinase inhibitors are under
development, and to date only SGI1776 has entered phase I
evaluation. However, its efficacy against multiple tumors and
lymphoma was limited, and the trial was terminated because
of cardiac toxicity (SuperGen press release November 10,
2010). Hence, PIM expression is a significant clinical problem
in lymphoma and a new therapeutic strategy is needed.
We identify a therapeutic strategy that is highly effective
against PIMexpressing lymphomas. Both the AKT and
PIM kinases control regulators of capdependent translation
Figure 4. The eIF4A helicase inhibitor silvestrol is active against mouse and human lymphomas irrespective of PIM expression. (A) Represen-
tative flow cytometry plots showing enrichment of subpopulations of Pim2-GFP–expressing Eµ-Myc/Tsc2/ upon treatment with vehicle, silvestrol,
rapamycin, or silvestrol and rapamycin in vitro. (B) Cumulative analysis of three separate experiments showing mean and SEM. (C) Eµ-Myc/Tsc2/
and Eµ-Myc/Tsc2//Pim2 cells, or 3T3 fibroblasts or VavP-Bcl2/Myc tumor cells were treated with indicated concentrations of silvestrol. Viability was
assessed after 24 h (mean and SEM of 4 separate assays per cell line). (D) Comparison of cell death induced by silvestrol or two PIM kinase inhibitors
(SGI-1776, SGI-1773) in a panel of human lymphoma cells (mean of three separate assessments and SEM). (E) Time to relapse in animals bearing
Eµ-Myc/Tsc2//Pim2 tumors that were treated with rapamycin (red; n = 9) or rapamycin and silvestrol (green; n = 9), or mice bearing parental
Eµ-Myc/Tsc2/ tumors treated with rapamycin (black dotted line; n = 9). (F and G) Immunoblot on human lymphoma cells Granta-519 (F) or DoHH2
and Su-DHL-10 (G) treated with vehicle (DMSO), the PIM inhibitor SG-1776, or silvestrol.
JEM Vol. 208, No. 9
Brief Definitive Report
In vivo treatment studies. Treatment studies with doxorubicin and/or
rapamycin were as previously described (Wendel et al., 2004; Mavrakis et al.,
2008). In brief, 106 primary lymphoma cells were injected into the tail vein
of 10–12wkold female C57BL/6 mice. Upon the formation of well
palpable tumors, the animals were treated with rapamycin (LC Laboratories;
4 mg/kg, i.p.), doxorubicin (SigmaAldrich; 10 mg/kg, i.p.), or a combina
tion of both. Eµ-Myc/Arf / tumors, which are homogeneous in respect to
p53 status, were used as controls where indicated. For treatment studies with
Eµ-Myc/Tsc2/ tumor cells, 10–12wkold female C57BL/6 mice were
injected with 250,000 tumor cells. Rapamycin was given as above, and silves
trol was dosed as previously described (Bordeleau et al., 2008), given at
0.2 mg/kg daily for 7 d. After treatment, the mice were monitored by palpa
tion and blood smears stained with Giemsa (Thermo Fisher Scientific).
Tumorfree and OS data were analyzed in the KaplanMeier format using
the logrank (MantelCox) test for statistical significance.
Cell culture, competition, and viability assays. Eµ-Myc/Tsc2/ and
Eµ-Myc/p53/ tumor cells were cultured in B cell media (1:1 DMEM/
IMDM, with 10% fetal bovine serum, penicillin/streptomycin, and lglutamine)
on feeder layers consisting of irradiated NIH3T3 cells. Competition assays
used the MSCVIRESGFP vector ± the indicated genes (Pim1, Pim2, and
eIF4E) or the shRNA vector MLP (Mavrakis et al., 2008) for shBad (see
below). GFP expression was assessed through FACS analysis (Guava EasyCyte;
Millipore). Experiments were repeated three or more times and averaged
based on fold change in the percentage of GFP+ cells before and after treat
ment with drug or vehicle. In competition time point experiments, cells were
treated with drug or vehicle on day 0 for 24 h and tracked for GFP expres
sion daily. Human lymphoma cell lines were cultured in RPMI1640 or
DME supplemented with 10% fetal bovine serum, penicillin/streptomycin, and
lglutamine. Cell viability was assessed with CellTiterGlo reagent (Promega).
IC50 values were determined from viability curves and represent a mean value
from 3–4 curves per cell line. The 4E-BP14A (in MSCVIRESGFP) vector
was a gift from the laboratory of N. Rosen (SloanKettering Institute, New
York, NY) and was sequence confirmed to contain mutation to alanine at resi
dues T37, T46, S65, and T70. Cytokine stimulation was performed for 6 or
12 h with 400 pg/ml recombinant mouse IL3 (Fitzgerald Industries) and
10 ng/ml recombinant mouse IL6 (Fitzgerald Industries). Puromycin selec
tions were performed for 2 d at a concentration of 2 µg/ml.
In vitro treatment studies. Rapamycin (LC Laboratories) was dissolved in
ethanol vehicle and stored as 10 mM stock solution protected from light at
20°C. It was diluted in icecold ethanol before use at the indicated concen
trations in the results and compared with 1:1,000 ethanoltreated vehicle
controls. Silvestrol was stored as 10mM stock solution in DMSO at 80°C
and diluted in DMSO before use at the indicated concentrations in the re
sults. SGI1773 and SGI1776 were provided by SuperGen Inc. and were
stored as 10mM stock solutions in DMSO at 20°C. Comparisons for sil
vestrol and the Pimkinase inhibitors were to 1:1,000 DMSOtreated vehicle
controls. For detecting drug effects by immunoblot, cells were treated with
10 nM rapamycin for 4 h, 10 nM silvestrol for 24 h, or 10 µM SGI1773
for 24 h.
Polysomal profiling. Sucrose density gradient centrifugation was used to
separate the ribosome fractions. 15 min before collection, cycloheximide
(100 µg/ml) was added to the culture medium. Cells were washed in icecold
PBS containing 100 µg/ml cycloheximide and harvested. Cell pellets were
resuspended in polysome lysis buffer (5 mM TrisHCl, pH 7.5, 2.5 mM
MgCl2, 1.5 mM KCl, 2 mM DTT, 0.5% Triton X100, 0.5% sodium deoxy
cholate, 100 µg/ml cycloheximide, RNAsin inhibitor, and protease and
phosphatase inhibitors). Cells were incubated on ice for 15 min, and then
centrifuged at 10,000 g for 10 min at 4°C. The supernatant (2 mg of protein)
was layered on a prechilled 10–50% linear sucrose gradient prepared in 5 mM
TrisHCl, pH 7.5, 2.5 mM MgCl2, and 1.5 mM KCl, and then centrifuged
in a Beckman SW41Ti rotor at 35,000 rpm for 2.5 h at 4°C. Gradients were
fractionated while monitoring absorbance continuously at 254 nm with a
effective against PIMexpressing human and mouse lym
phoma cells and far superior to current PIM kinase inhibitors.
Therapeutic blockade of translation affects several shortlived
oncoproteins, including the PIM1/2 kinases and c-MYC,
MCL1, and Cyclin D1. Silvestrol does not cause the feedback
activation of upstream signaling molecules that has been seen
upon rapamycin treatment (O’Reilly et al., 2006). In sum
mary, PIM kinase expression adversely affects outcomes in
NHL, and targeting the translation of oncoproteins like PIM
and cMyc effectively disables this critical output of converg
ing oncogenic pathways.
MATERIALS AND METHODS
TMAs. TMAs were constructed from paraffinembedded tumor cores of
452 NHL patients treated at MSKCC since the mid1980s (173 DLBCL,
205 FL, 37 MCL, and 37 CLL/SLL). Use of tissue samples was approved by
the Institutional Review Board and the Human Biospecimen Utilization
Committee. All cancer biopsies were evaluated at MSKCC, and the histolog
ical diagnosis was based on hematoxylin and eosin (H&E) staining. TMAs
were constructed, stained, and scored as previously described (Hedvat et al.,
2002) with antibodies against Pim1 and Pim2 (Cell Signaling Technology).
Pim1 polyclonal antibody staining was performed at 1:100 dilution using the
manufacturer’s protocol, with secondary staining by OmniMap DAB anti
Rb Detection kit (Ventana). Pim2 monoclonal antibody staining was per
formed manually at 1:100 dilution in citric acid, pH 6, with rabbit secondary
antibody and finished with DAB (3,3Diaminobenzidine). All TMA scoring
was performed by an expert lymphoma hematopathologist.
Clinical data and analyses. Under MSKCC IRB waiver approval, clinical
data were collected on patients whose tumors appear on the DLBCL and
FL TMAs. Of the FL cases, we identified 66 whose disease required treat
ment, whose specimen on the TMA was from before their initial therapy, and
for whom treatment data and Pim scores were available. These cases were
subjected to KaplanMeier TTE and OS analyses from initiation of therapy
and date of diagnosis, respectively. Events were defined as progression of dis
ease, death, or secondary malignancy. Logrank analysis was used to compare
groups. The same analyses were performed on 116 DLBCL patients with
available treatment data and whose biopsy sample on the TMA was from be
fore initial therapy. PIM+ versus PIM patient groups were compared for
age, sex, and additional clinical variables listed in Tables S1 and S2 based on
data availability. 2 or fisher’s exact test was used to compare categorical variables
and Wilcoxon ranksum test was used to compare continuous variables.
Mouse lymphoma generation and analysis. All animal experiments
were approved by the MSKCC Institutional Animal Care and Use Committee
in compliance with the U.S. Department of Health and Human Services Guide
for the Care and Use of Laboratory Animals. The Eµ-Myc model of aggressive
lymphoma (Adams et al., 1985) and the VavPBcl2 model of follicular lym
phoma (Egle et al., 2004) were adapted to the transplantation approach using
retrovirally transduced HPCs (Wendel et al., 2004). In brief, we isolated
HPCs from the fetal livers of day 13.5–14.5 transgenic embryos and infected
them with retroviral constructs coexpressing GFP and murine Pim2 or con
stitutively active myristoylated AKT using the MSCVIRESGFP vector.
The HPCs were then transplanted into syngeneic wildtype C57/B6 recipient
animals after sublethal irradiation (same day 4.5G + 4.5G). We then tracked
animals for tumor onset by observation, palpation, and blood smear evalua
tion. Disease onset data were subjected to KaplanMeier analysis and the log
rank (MantelCox) test for statistical significance. H&E, Ki67, TUNEL,
phosphoAKT, phospho4EBP1, phosphoS6, Pim2, and surface marker analy
sis were previously described (Mavrakis et al., 2008). Eµ-Myc/Tsc2/ lym
phomas are generated by crossing Eµ-Myc+/ mice to Tsc2+/ mice (Mills et al.,
2008). Double heterozygous offspring generate B cell tumors because of loss of
heterozygosity at the Tsc2 locus, resulting in tumors that can be cultured ex vivo.
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Western blot analysis. Immunoblots were performed from wholecell
lysates. In brief, 20–50 µg of protein/sample were resolved on SDSPAGE
gels and transferred to ImmobilonP membranes (Millipore). Antibodies
were against human PIM1, human PIM2, AKT, phosphoAKT (S473), ribo
somal protein S6, phosphorpS6 (S240/244), 4EBP1, phospho4EBP1 (S65),
eIF4E, phosphoeIF4E (S209), BAD, phosphoBAD (S112), phosphoMDM2
(S166), CyclinD1 (Cell Signaling Technology); mouse Pim1, mouse Pim2, c-Myc
(Santa Cruz Biotechnology); MCL1 (Rockland Immunochemicals); and
shRNA gene knockdown. RNAi knockdown of mouse Bad was per
formed as previously described (Mavrakis et al., 2008) using the GFP
coexpressing, puromycinselectable shRNA vector MLP. Three potential
shRNAs were generated and tested by infecting FL512 cells with them
or empty MLP vector, purification through puromycin selection, and
immunoblotting protein lysates for Bad protein levels. Sequences of the
hairpins were as follows: #1, 5TGCTGTTGACAGTGAGCGACAG
CACTAGCGTCTTCCTGCTGCCTACTGCCTCGGA–3; #2, 5TGCT
TGCCTCGGA3; #3, 5TGCTGTTGACAGTGAGCGACTGCAA
Online supplemental material. Table S1 shows complete TMA scoring.
Tables S2 and S3 show clinical characteristics of analyzed FL and DLBCL pa
tients, respectively. Tables S4 and S5 show statistical analyses of FL and DLBCL
patients, respectively, by PIM1 and PIM2 expression. Online supplemental mate
rial is available at http://www.jem.org/cgi/content/full/jem.20110846/DC1.
We thank W. Tam, J. Chaudhuri, R. Gascoyne, and SuperGen, Inc. for reagents.
This work is supported by grants from the National Cancer Institute (R01-
CA142798-01), the AIDS Malignancy Consrtium, a P30 supplemental award (H.G.
Wendel), the Leukemia Research Foundation (H.G. Wendel), the Louis V. Gerstner
Foundation (H.G. Wendel), the WLBH Foundation (H.G. Wendel), the Society of
MSKCC (H.G. Wendel), the Starr Cancer Consortium grant I4-A410 (H.G. Wendel),
the Charles A. Dana Foundation (J.H. Schatz), the Lymphoma Research Foundation
(J.H. Schatz), the ASCO Cancer Foundation (J.H. Schatz), the MSKCC Translational-
Integrative Medicine Research Fund (J.H. Schatz), the Lacher Foundation (J.H.
Schatz), Canadian Cancer Society Research Institute (CCSRI #20066 to J. Pelletier),
and National Institutes of Health (GM-073855 to JAP Jr.).
Author contributions: J.H. Schatz and E. Oricchio conducted experiments and were
involved in the experimental design and data analysis; A.L. Wolfe, N.C. Pagano, R.K.
Vinagolu., M. Jiang, I. Linkov, and J. Maragulia assisted with experiments and data
collection; W. Shi and Z. Zhang analyzed clinical data; A.D. Zelenetz and N. Rosen
analyzed data and helped with study design; J.A. Porco, Jr. synthesized silvestrol
and provided information on its use; J. Teruya-Feldstein performed pathological
analyses including TMA scoring; J. Pelletier provided silvestrol, analyzed data, and
edited the manuscript; J.H. Schatz and H.G. Wendel wrote the paper; and H.G.
Wendel designed the study.
Author information The authors declare no competing financial interests.
Submitted: 28 April 2011
Accepted: 27 July 2011
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