Regular Article

LYMPHOID NEOPLASIA

OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane

localization and activation of chronic lymphocytic leukemia cells
Ta-Ming Liu,1 Yonghua Ling,2 Jennifer A. Woyach,1 Kyle Beckwith,1 Yuh-Ying Yeh,1 Erin Hertlein,1 Xiaoli Zhang,3
Amy Lehman,3 Farrukh Awan,1 Jeffrey A. Jones,1 Leslie A. Andritsos,1 Kami Maddocks,1 Jessica MacMurray,1
Santosh B. Salunke,4 Ching-Shih Chen,1,5 Mitch A. Phelps,4 John C. Byrd,1,5 and Amy J. Johnson1,5
1
Department of Internal Medicine, Division of Hematology, Wexner Medical Center, 2Pharmacoanalytical Shared Resource, Comprehensive Cancer Center,
3
Center for Biostatistics, 4Department of Pharmaceutics and Pharmaceutical Chemistry, and 5Department of Medical Chemistry and Pharmacognosy,
College of Pharmacy, The Ohio State University, Columbus, OH

Aberrant regulation of endogenous survival pathways plays a major role in progression

Key Points

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of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors
• OSU-T315 impedes AKT within the tumor environmental niche activates survival and proliferation pathways in
localization in lipid rafts. CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway ap-
• OSU-T315 shows in vitro and pears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting
in vivo therapeutic effects. this axis have shown clinical activity. Here we investigate OSU-T315, a compound that dis-
rupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by
OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful
Bruton’s tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly
abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases.
Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like
receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active
drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of
OSU-T315 with potential therapeutic application in CLL. (Blood. 2015;125(2):284-295)

Introduction
Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in
adults and remains incurable despite the introduction of targeted agents.
CLL also has an uncertain etiology,1,2 although current data support
that CLL originates from antigen-experienced, postgerminal center
B cells.3 CLL has multiple recurrent cytogenetic abnormalities in-
cluding del(13q14.3), trisomy 12, del(11q22.3), and del(17p13.1),
of which the latter 2 portend a more rapid disease progression and
shorter survival from diagnosis.4 Approximately 60% to 65% of
CLL cases exhibit somatic hypermutation in immunoglobulin heavy
chain variable (IGHV) genes (M-CLL), whereas 35% to 40% of CLL
cases are categorized with unmutated IGHV status (U-CLL), which
is associated with poor prognosis.5,6 The U-CLL patient subset also
has a high proportion of ZAP-70 expression,7 enhanced B-cell receptor
(BCR) signaling, and a disproportionate number of del(11q22.3) and
del(17p13.1) cases. Overall, identification of biological markers
associated with clinical outcome facilitates the identification of
therapies targeted toward aberrant signaling pathways.
The current initial therapy for CLL patients lacking del(17p13.1)
typically includes fludarabine and cyclophosphamide plus rituximab
for younger, fit patients,8 whereas for older or infirm patients,
chlorambucil plus obinutuzumab9 is most appropriate. Patients with
del(17p13.1) do not benefit in terms of progression-free survival and
overall survival with chemoimmunotherapy.10,11 Despite chemoimmu-
notherapy prolonging survival, this treatment is not curative. A proposed
reason that available CLL therapies are incompletely effective is the
increased proliferation and acquisition of tumor cell resistance to ap-
optosis as a result of stimuli within microenvironment of lymphoid
tissues. Following recent advances in our understanding of CLL disease
biology, efforts have focused on antagonizing oncogenic signaling ini-
tiated from the tumor microenvironment.12 Key prosurvival signals in
CLL include BCR activation,13-15 the tumor necrosis factor receptor
family molecules CD40L, B-cell activating factor, and a proliferation-
inducing ligand,16,17 and the chemokines C-C motif ligand (CCL)-3,
CCL4,18 C–X–C motif ligand (CXCL)-12,19 and CXCL13,20 all of
which augment downstream activation of protein kinase B (AKT)
and/or extracellular signal-regulated kinase (ERK) signaling in CLL
cells and contribute to CLL survival and proliferation.21 To date, the
best success in targeting the pathways activated by these signals
has been through the use of agents inhibiting proximal or distal
BCR signaling, such as the phosphoinositide 3-kinase (PI3K) p110d
inhibitor idelalisib22 and the Bruton’s tyrosine kinase (BTK) inhibitor
ibrutinib.23 Despite a high frequency of durable partial responses with
these agents in CLL patients, complete remissions are infrequent.
Indeed, none of these agents sufficiently overcomes AKT and/or

Submitted June 23, 2014; accepted September 18, 2014. Prepublished online
as Blood First Edition paper, October 7, 2014; DOI 10.1182/blood-2014-06-
583518.
M.A.P., J.C.B., and A.J.J. contributed equally to this study.
There is an Inside Blood Commentary on this article in this issue.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.

The online version of this article contains a data supplement. © 2015 by The American Society of Hematology

284 BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 OSU-T315 ANTAGONIZES AKT LOCALIZATION IN RAFTS 285

Table 1. CLL patients The activation of the PI3K/AKT pathway is initiated at the
ID 13q14.3 11q22.3 17p13.1 trisomy 12 IgVH plasma membrane, where phosphatidylinositol (3,4,5) trisphosphate
CLL 0083 V — — — M 7.6% (PIP3) generated by PI3 kinase recruits AKT to the unique mem-
CLL 0141 V — — — M 2.1% brane compartments termed lipid rafts on interaction via Pleckstrin
CLL 1330 V — — — U 1.0% homology domains, leading to its subsequent phosphorylation and
CLL 1227 V V — — U 0% activation by phosphoinositide-dependent kinase-1 (PDK)-1/2.24
CLL 0588 — V — — U 0% Recent studies show that these glycosphingolipid- and cholesterol-
CLL 0801 V V — — U 0%
rich rafts serve as platforms for the initiation of a variety of signaling
CLL 1201 — V — — U 0%
pathways.25 These include the PI3K/AKT,26 CD40L,27 and BCR28
CLL 0810 — — — — U 0%
CLL 1529 V — — — M 8.8%
signaling pathways, each of which are associated with CLL tumor
CLL 1600 V — — — U 0% cell survival and disease progression. Notably, disruption of lipid
CLL 0881 — V — V N/A rafts by cholesterol sequestration using saponin, cholesterol depletion
CLL 1461 — — — — U 0.7% by methyl-b-cyclodextrin,29 or inhibition of cholesterol biosynthesis
CLL 0166 — — — — U 0% by simvastatin30 results in ablation of AKT phosphorylation and
CLL 0742 — — — V U 0% induces preferential cytotoxicity toward malignant cells. 31 Like-
CLL 1258 — — V — U 0.7%
wise, investigations using alkyl-lysophospholipid analogs fur-

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CLL 1766 V V V — N/A
CLL 1593 V — V — N/A
ther support the antitumor potential of targeting lipid rafts in
CLL 1446 — V V V N/A mantle cell lymphoma and CLL via the recruitment of Fas/CD95.32
CLL 0170 V — V — M 10% Building on these promising results, we herein report that the
CLL 0222 V — V — U 0% novel agent OSU-T315, modified from the scaffold of the AKT
CLL 1606 V — V — U 0.3% binding site at the integrin-linked kinase (ILK), uses an analogous
—, negative; M, mutated; N/A, not applicable; U, unmutated; V, positive.
mechanism of antitumor activity in CLL by displacing AKT from
lipid rafts.
ERK signaling pathways concurrently in the presence of multiple ILK is a master regulator of intracellular signaling that con-
survival stimuli. Agents that inhibit AKT and/or ERK pathways in a trols cell proliferation, survival, migration, invasion, and angiogenesis.33
novel manner therefore represent an exciting strategy for this disease. The crucial function of ILK in tumorigenesis involves activation of

Figure 1. OSU-T315 induces preferential cytotoxicity

in CLL cells. 1E7/mL primary CLL cells from patients;
(A) healthy B cells (CD191) and (B) T cells (CD31) from
leukopaks purified by the CD191 or CD31 enrichment
kit, respectively, were incubated in complete RPMI with
10% fetal bovine serum followed by increasing dose of
OSU-T315 treatment. Cells viability was analyzed by
flow cytometry at 24 hours. (C) The response toward
OSU-T315 in CLL with cytogenetic abnormalities
were analyzed by del(17p13.1) and IGVH status or
(D) del(11q22.3) and del(13q14.3). (E) 1E6/mL cells in
complete RPMI with 10% fetal bovine serum was in-
cubated and treated with increasing concentration of
OSU-T315. Cells viability on treatment in Mec-1 or
OSU-CLL cells was examined at 24 hours by Annexin V/PI
staining. (F) Three ibrutinib-resistant samples were treated
with OSU-T315 for 24 hours in comparison with non-
resistant CLL cells; 1 mM ibrutinib was used for treatment
for 30 minutes and then washed out; 1 mM CAL-101 was
used for treatment for 24 hours.
286 LIU et al
AKT via Ser473 phosphorylation, suggesting ILK as a promising
target in malignancies that rely on the PI3K pathway.34 Similarly,
overexpression of ILK in myoblasts results in enhanced ERK acti-
vation, supporting the involvement of ILK in the mitogen-activated
protein kinase (MAPK) pathway.35 Recently, generation of the novel
ILK inhibitor OSU-T315 was reported.36 Given the justification that
targeted therapy directed at ILK would be effective in CLL through
suppressing AKT and/or ERK activation, we pursued preclinical in vi-
tro and in vivo studies to determine the activity of OSU-T315 toward
primary CLL cells. Concurrently, we demonstrate that this agent
caused disruption of PI3K and MAPK signaling promoted by
BCR or integrin engagement, besides specific inhibition of PI3K
axis mediated by CD40 and TLR9. Remarkably, we verify that
these effects are not through inhibition of ILK as expected, but rather
by the prevention of AKT recruitment to lipid rafts. This surprising
finding introduces a novel mechanism to therapeutically target
CLL.
BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2
Figure 2. The apoptotic machineries were induced
on T315 treatment. (A) CLL cells were incubated in
a-IgM coated plates or (C) fibronectin-coated plates or
1 mg/mL VCAM-1 with vehicle or OSU-T315 for 16 hours.
Cell lysate was analyzed by immunoblotting and nor-
malized to GAPDH, and the ratio of densitometry to
GAPDH was shown beneath the blots. (B) Mcl-1 expres-
sion level was quantified according to the results of
immunoblotting in the separated conditions of plate-
coated a-IgM, 0.5 mg/mL CD40L, 3.2 mM CpG-ODN,
or (D) fibronectin-coated plates or 1 mg/mL VCAM-1
along with the treatment of vehicle or 4 mM OSU-T315
for 16 hours. (E) Caspase 3/7 activity was accessed by
Caspase-Glo 3/7 Assay Systems (Promega) and normal-
ized to vehicle after 16-hour treatment; 20 mM of Q-VD-
OPh served as the negative control, whereas 500 nM
flavopiridol served as the positive control. (F) Mec-1
or OSU-CLL cells pretreated with vehicle or 20 mM
z-VAD-FMK prior to 4 mM OSU-T315 treatment were
analyzed by Annexin V/PI staining after 24 hours.
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Materials and methods
Reagents and antibodies
OSU-T315 was synthesized as previously described.36 OSU-arg was ac-
quired from the National Cancer Institute (Bethesda, MD). Stock solutions
were prepared in dimethylsulfoxide. The commercial sources of antibodies
used are listed in the supplemental Methods on the Blood Web site.
CLL, normal B-cell, and T-cell isolation
Peripheral blood samples were obtained from CLL patients after written
informed consent was provided on an The Ohio State University institu-
tional review board-approved protocol. Research was conducted in ac-
cordance with the Declaration of Helsinki. Human CLL and normal B or
T cells were isolated and cultured as previously described23 and specified in
the supplemental Methods.
BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 OSU-T315 ANTAGONIZES AKT LOCALIZATION IN RAFTS 287

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Figure 3. OSU-T315 targets intrinsic AKT and ERK signals cascades in CLL cells. (A) Lysate from Mec-1 and OSU-CLL cells treated with serial concentrations of OSU-
T315 for 15 minutes are subjected to western blot analysis. (B) Lysate from primary CLL cells treated with OSU-T315 were subjected to analyze PDK1 (Ser241) and
subsequent AKT (Thr308) activation. (C) In vitro kinase activity of class I PI3K is evaluated by the PI3 Kinase Activity/Inhibitor Assay Kit (Millipore) according to the instruction
manual; 100 nM Wortmannin was applied as positive control. The biotinylated-PIP3 was set as 100%. The kinase reactions with vehicle or OSU-T315 were referenced to the
biotinylated-PIP3 signal to have the relative percentage of inhibition. (D) RAS activity in 697 cells on treatments was measured by the Active Ras Detection Kit (Cell Signaling)
according to the instruction manual. Guanosine triphosphate gS (positive control) and guanosine diphosphate (negative control) ensured the immunoprecipitation procedures
worked properly, whereas insulin-like growth factor-1 served as a positive control to activate Ras. (E) Mec-1 and OSU-CLL cells pretreated with Okadaic acid (1 mM)
were incubated with either OSU-T315 (4 mM) or OSU-03012 (5 mM), the PDK1 inhibitor, and the total lysate was subjected to immunoblotting to verify downstream
signaling.

Cell lysis and immunoblot
Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce).
Proteins were quantified by the BCA protein assay kit (Pierce) and separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots
were probed with the appropriate primary and horseradish peroxidase-
conjugated secondary antibodies and developed with chemiluminescent
substrate (Pierce), followed by detection using X-ray film and quantifica-
tion using ImageJ software.
Assessment of cell death
Cell viability was assessed using annexin-V and propidium iodide (PI)
double staining followed by analysis on an EPICS XL flow cytometer
288 LIU et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

Table 2. Densitometry for western blots for Figure 3A

Mec-1 OSU-CLL
C 1 mM 2 mM 3 mM 4 mM C 1 mM 2 mM 3 mM 4 mM
P-FAKTyr397 6.5 8.2 6.2 5.1 5.0 2.2 1.9 2.4 2.6 2.4
P-FAKTyr925 4.2 3.6 4.7 4.0 3.5 2.8 3.5 4.3 3.7 3.2
P-BTK 5.0 6.2 6.2 5.8 5.7 7.5 7.7 9.1 9.1 8.5
P-AKTSer473 11.1 6.5 3.2 1.9 1.8 15.1 10.0 6.6 3.1 2.3
P-FAKThr308 13.6 6.9 7.1 3.7 2.8 14.3 6.5 3.6 3.3 2.4
P-Craf 17.6 16.7 11.7 8.1 6.4 14.0 9.6 8.4 6.4 6.2
P-ERK 15.9 7.5 2.9 2.6 2.6 12.6 6.5 7.7 7.4 7.5
P-P38 8.4 8.1 7.1 5.7 4.5 5.6 6.4 7.0 5.8 4.1
P-GSK3b 6.3 5.2 4.8 4.1 3.0 6.9 4.9 3.9 3.3 3.6
P-PDK1 4.3 5.0 5.4 5.0 4.3 4.3 5.0 6.6 5.8 5.2

(Beckman-Coulter). Mitochondrial activity was measured by the CellTiter comparable efficacy toward CLL cells from ibrutinib-resistant
96 AQueous One Solution Cell Proliferation Assay (Promega) according patients (Figure 1F).

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to the manufacturer’s instructions.

Animal studies
CD191 cells were obtained from spleens of TCL1 transgenic mice with CLL-
like leukemia and splenomegaly, and 1 3 106 cells were injected intravenously
into a C57BL/6 mouse as previously described37 and specified in the sup-
plemental Methods.
Statistical analysis
Percent viability of CLL compared with B or T cells on treatment was
outlined by means with standard error of the mean and analyzed based on
unpaired Student t tests. The ratios of phospho-protein were measured after
normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
expression. Differences between treatments were calculated by unpaired
Student t tests. Overlap index of AKT content with lipid rafts was analyzed
using Olympus FluoView viewer. For mouse models, overall survival was
obtained using the Kaplan-Meier method. All tests were 2 sided, and
statistical significance was declared for P , .05.
Results
OSU-T315 induces selective cytotoxicity toward CLL cells
We first sought to assess the preclinical efficacy of OSU-T315
toward primary CLL cells. Our data indicate dose-dependent
cytotoxicity toward CLL cells. The LC50 after 24-hour in-
cubation of primary CLL (n 5 21; Table 1) cells was 4.83 6 0.83 mM.
Contrasting with this, the cytotoxicity of OSU-T315 in normal
B (n 5 7) or T cells (n 5 8) was significantly lower (LC50 . 10 mM;
P , .0001 for each; Figure 1A-B), suggesting a therapeutic index of
OSU-T315. CLL acquiring genomic aberrations including high-
risk features del(17p13.1), unmutated IGVH, or del(13q14.3) and
del(11q22.3) also reveal undifferentiated sensitivity toward OSU-
T315 (Figure 1C-D). We next examined the effect of T315 toward
2 CLL-derived cell lines, Mec-1 and OSU-CLL (Figure 1E), and
found similar dose-dependent cytotoxicity (LC50 of 2-3 mM in
both) after 24-hour treatment. Remarkably, OSU-T315 exhibits
OSU-T315 reduces Mcl-1 and Bcl-xl levels to trigger caspase
activation in CLL cells
Bcl-2 family proteins are known to modulate apoptosis in CLL.
Among these family members, Mcl-121,38 appears to play a partic-
ularly important role in CLL cell survival and can be upregulated by
BCR activation.39 To characterize the effect of OSU-T315 on Bcl-2
family proteins, protein expression of Mcl-1, Bcl-2, Bad, and Bim
was examined. Although expression levels of Bcl-2, Bad, and Bim
were unaffected, Mcl-1 levels were increased upon BCR, TLR9, and
CD49d activation, and OSU-T315 treatment was able to com-
pletely reverse this effect (Figure 2A-D). Remarkably, Mcl-1-
overexpressing cells, which show resistance to fludarabine, were
similarly sensitive to OSU-T315, indicating that OSU-T315
activity appears to be independent of Mcl-1, unlike the majority
of currently available CLL agents (supplemental Figure 3A). In-
terestingly, Bcl-xl upregulation mediated by cytosine guanine
dinucleotide (CpG) or CD40L was strongly repressed by OSU-T315
(supplemental Figure 3A). Because Mcl-1 or Bcl-xl is involved in
antagonizing apoptosis in CLL,21,39 our results further suggest that
caspase 3/7 activity is significantly augmented when increasing
concentrations of OSU-T315 are administrated (Figure 2E). The
importance of caspase activation in OSU-T315-mediated apoptosis
was verified using Z-VAD-FMK, a pan-caspase inhibitor (Figure 2F).
These data demonstrate that OSU-T315 mediates cytotoxicity in CLL
or CLL-like cell lines in a caspase-dependent manner independent
of Mcl-1 protein expression.
OSU-T315 inhibits AKT signaling independent of ILK
Activation of the PI3K/AKT pathway is essential for CLL survival
and proliferation.21 Given that OSU-T315 has been shown to inhibit
activation of AKT and ERK through targeting ILK in solid tumors,36
components of key signaling pathways were evaluated following
OSU-T315 treatment in Mec-1 and OSU-CLL cells (Figure 3A and
Table 2). Consistent with the previous report, this cellular profiling
demonstrates notably diminished AKT and ERK phosphorylation on

Table 3. Densitometry for western blots for Figure 3B

PT1 PT2 PT3 PT4
C a-IgM a-IgM1T315 C a-IgM a-IgM1T315 C a-IgM a-IgM1T315 C a-IgM a-IgM1T315
P-PDK1 5.8 4.2 5.0 6.8 5.1 5.6 9.2 8.2 8.1 5.7 4.4 4.3
P-AKTThr308 2.0 3.3 2.1 2.4 5.1 2.1 2.1 7.2 2.0 2.5 5.9 3.6
BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 OSU-T315 ANTAGONIZES AKT LOCALIZATION IN RAFTS 289

Table 4. Densitometry for western blots for Figure 3E

Mec-1 OSU-CLL
C T315 Okadaic acid T3151Oka OSU-03012 OSU-030121Oka C T315 Okadaic acid T3151Oka OSU-03012 OSU-030121Oka
P-Craf 12.8 5.9 15.0 7.0 10.5 11.9 10.4 5.8 12.7 6.6 7.1 9.3
P-AKTSer473 6.0 3.1 8.9 3.1 3.9 6.6 9.7 3.3 10.0 3.1 6.8 8.9
P-FAKThr308 8.7 5.7 12.6 6.3 6.2 10.6 8.1 5.6 11.4 5.6 6.5 9.8
P-GSK3b 6.1 4.4 8.3 6.4 4.8 7.7 4.1 2.7 8.4 5.3 4.1 7.0
P-ERK 15.9 3.4 21.5 7.0 15.8 20.0 14.2 5.5 19.6 8.1 10.8 16.9

treatment, along with dephosphorylation of glycogen synthase
kinase-3 beta (GSK-3b), a direct downstream target of AKT.40 To
elucidate the role of ILK in phosphorylating Ser473 site of AKT, we
silenced ILK by short hairpin RNA. Despite efficient knockdown of
ILK (supplemental Figure 1A), the proliferation rate in Mec-1 cells
activity of PI3K, PDK1, RAS, or PP2A, we postulated that OSU-
T315 may elicit its effects through altering recruitment of AKT
to lipid rafts. To determine whether OSU-T315 impedes the re-
cruitment of AKT, myristoylated-AKT (myr-AKT) that sponta-
neously translocates to lipid rafts42 was introduced into Mec-1 cells

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was not altered (supplemental Figure 1B). Furthermore, ILK-depleted
cells showed no differences in phosphorylation of AKT Ser473 or ERK,
either at baseline or following anti-IgM (supplemental Figure 1C)
or fibronectin (supplemental Figure 1D) stimulation. ILK down-
regulation also did not abrogate OSU-T315–mediated killing (sup-
plemental Figure 1E). Additionally, the interaction between AKT and
ILK is not affected (supplemental Figure 1F). Together, these results
suggest a cytotoxic role of OSU-T315 independent of ILK in
lymphoid leukemia cells.
OSU-T315 does not influence proximal membrane signaling
of AKT
Given that the localization of PDK1 to plasma membrane is essential
to phosphorylate AKT and the generation of PIP3 via PI3-kinase is
essential for this recruitment, we hypothesized that OSU-T315 may
interfere with PDK1 activity or even proximal PI3K. To investigate
these possibilities, we examined the effects of OSU-T315 on AKT-
related signaling events. OSU-T315 treatment failed to disrupt
PDK1 phosphorylation41 (Figure 3B and Table 3), PI3 kinase activity
(Figure 3C), or Ras activity regarding the interaction with proto-
oncogene serine/threonine-protein kinase (Figure 3D) under conditions
in which AKT phosphorylation is inhibited. Other membrane-
associated kinases including focal adhesion kinase (FAK) and BTK,
a key component of the BCR signaling pathway, were also not af-
fected by OSU-T315 (Figure 3A). Similarly, the activation of
phosphatase PP2A, which has been known to negatively regulate
AKT activity, was not influenced by T315 as evidenced by continued
dephosphorylation of AKT in the presence of okadaic acid, the PP2A
inhibitor (Figure 3E and Table 4). Together, these findings indicate
that OSU-T315 induces cytotoxicity in CLL cells by suppressing
intrinsic AKT activation without influencing proximal kinases or
relevant phosphatases.
to reinforce membrane docking of AKT in resting cells (Figure 4A).
The diminished phosphorylation of myr-AKT was observed fol-
lowing OSU-T315 treatment (Figure 4B and Table 5), in parallel
with reduced content of myr-AKT within the lipid raft fraction
(Figure 4C-D and Table 6) while retaining equivalent amounts of
PI3 kinases, phosphatase and tensin homolog (PTEN), and BCR-
associated molecules Lyn and BTK. These data demonstrate that
OSU-T315 inhibits AKT translocation into lipid rafts without af-
fecting raft integrity. Furthermore, confocal microscopy to examine
protein distribution shows reduced colocalization of myr-AKT
with lipid rafts on OSU-T315 treatment (Figure 4E-F). Consistently,
a-IgM-induced AKT translocation is abrogated by OSU-T315
(Figure 4G). Together, these findings confirm that OSU-T315
inhibits AKT activation by preventing its localization into lipid
rafts.
BCR-, CD40-, and Toll-like receptor 9-induced survival signals
are abrogated by OSU-T315
Diverse stimuli within tumor environments provoke ATK activa-
tion. Numerous studies indicate that the BCR is a key transmitter
of survival signals in CLL cells.14 On engagement of BCR, PI3K
and MAPK cascades are activated, from which CLL acquires the
competency to evade apoptosis. To assess whether OSU-T315 can
overcome external survival stimuli, CLL cells stimulated with im-
mobilized a-IgM, CD40L, or CpG were treated with different
concentrations of OSU-T315. The results suggest the potency of
OSU-T315 suppressing downstream AKT activation triggered by
external factors (Figure 5A-B). The protection of CLL cells mediated
by these external stimuli was abrogated by increasing doses of
OSU-T315 (Figure 5E-G).
CD49d-mediated survival signals are abolished by OSU-T315

OSU-T315 antagonizes AKT membrane localization CLL cells traffic and interact with stromal or nurse-like cells
within lymphoid tissues through the VLA-4 (a4b1, CD49d/
It is now evident that lipid rafts are highly important for the inter- CD29) complex, from which survival signals originate to pre-
actions and regulations of membrane-localized kinases, crucially vent spontaneous or drug-induced apoptosis in part resulting
mediating AKT translocation and downstream activation.30,31 As from AKT activation.43,44 Given that OSU-T315 interferes with
OSU-T315 inhibits AKT phosphorylation without affecting the integrin signaling as evidenced by inhibition of fibronectin-
induced AKT phosphorylation, we examined the potential of OSU-
Table 5. Densitometry for western blots for Figure 4B T315 to block CD49d-mediated protection of CLL cells. Our results
demonstrate CD49d activation via interaction with fibronectin or
C 1 mM 2 mM 3 mM 4 mM 5 mM
vascular cell adhesion molecule (VCAM)-1 can promote both AKT
P-AKT 6.1 3.7 3.6 3.6 3.3 3.1
and ERK signals, and these effects are suppressed by OSU-T315
P-ERK 9.7 6.5 5.6 4.8 5.5 6.7
(Figure 5C-D).
290 LIU et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

Table 6. Densitometry for western blots for Figure 4C OSU-T315 demonstrates potential therapeutic implications in high-
C T315 C T315 C T315 risk patients with del(17p13.1) or unmutated IGVH, as well as ibrutinib-
AKT 5.1 3.9 7.5 5.5 4.7 3.1 resistant patients. We further clarify in vivo efficacy showing improved
BTK 2.2 4.1 4.8 5.6 3.3 3.8 median survival in a TCL-1 transplant model, along with selective
LYN 8.6 9.7 5.6 6.7 6.1 5.1 targeting of CLL cells relative to normal B or T lymphocytes.
P13K a 5.7 5.0 4.9 5.2 3.3 4.1 Targeting BCR-associated kinases with small compounds has
P13K d 8.3 10.6 8.0 7.4 2.7 5.4 emerged as an exciting new treatment paradigm in CLL.47 The shift
PTEN 5.2 6.5 6.5 6.9 3.8 5.5 of focus toward BCR-associated kinases is owed in part to a better
understanding of the essential role of the BCR in CLL pathogenesis
OSU-T315 has in vivo activity in the TCL1 mouse model of and the promising clinical results achieved with continuous dosing
human CLL of BTK and PI3K inhibitors. Notably, all agents effective in targeting
BCR signaling suppress the PI3K/AKT signaling cascade. Unusual
Em-TCL1 adult mice develop clonal B-cell leukemia with surface activation of AKT drives CLL expansion and evades apoptotic
expression of immunoglobulin (IgM)1/CD191/CD51,45 exhibiting mechanisms, critically contributing to this lymphoproliferative
features similar to human CLL cells with regard to activation of AKT disorder.48 Previous studies identified the importance of the PI3K d
signaling, in part resulting from the interaction of TCL1 with AKT subunit in provoking AKT activation in CLL cells, thus driving the
and subsequent enhancement of its kinase activity.46 Thus, leukemia

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cells derived from Eu-TCL1 mice were transplanted into C57BL/6
mice to assess the in vivo efficacy of OSU-T315. Mice that de-
veloped leukemia in the bloodstream ($10% CD51/CD191 cells)
were treated orally with vehicle or OSU-T315 (50 mg/kg daily).
Mice that received OSU-T315 show significantly reduced white
blood cell (WBC) counts after 4 weeks of treatment (P 5 .036; n 5 6
per group; Figure 6A). This improvement also correlated with
prolonged overall survival in the OSU-T315-treated group (P 5 .037;
n 5 6 per group; Figure 6B). Improved in vivo efficacy of OSU-
T315 was further validated by intraperitoneal delivery approach
(P 5 .012; n 5 9 per group; Figure 6C). Collectively, these findings
show that OSU-T315 displays the capacity to delay leukemia pro-
gression and significantly increase overall survival in mice bearing
TCL1 leukemia cells.
Pharmacokinetic properties of OSU-T315
Previous studies verified the efficacy of orally administered OSU-
T315 in mice xenografted with prostate cancer cells.36 Although our
data also confirm the in vivo efficacy of OSU-T315 in leukemia,
pharmacokinetic studies on OSU-T315 remain necessary to optimize
the treatment regimen. Therefore, plasma from mice treated with
OSU-T315 by oral gavage or intravenous or intraperitoneal injection
was analyzed (Figure 6D and Table 7). Results indicate a simi-
lar elimination half-life by these delivery approaches. Although
intravenous administration achieves peak concentration of OSU-
T315 within 5 minutes, either intraperitoneal or oral administration
requires 2 hours to reach maximum levels in systemic circulation,
and these maximum levels are substantially lower than the value
achieved via the intravenous route, which may relate to less favorable
absorption, distribution, metabolism, and excretion properties
of OSU-T315 in existing formulation. Importantly, the low bio-
availability (5.8%) via the oral route may in part have contributed to
the relatively modest survival benefit of this agent, and strategies are
being explored to address this obstacle.
development of Idelalisib (GS-1101)49 and IPI-145 (INK-1147)50
that both show significant clinical activity through abrogating the
PI3K/AKT axis. Phosphorylation of AKT initially occurs at the
Thr308 site in the activation loop by PDK1, with full activation
occurring following phosphorylation of Ser473 in the C-terminal
hydrophobic domain by the putative PDK2 kinase. In contrast to
existing agents targeting upstream kinases, OSU-T315 impedes the
recruitment of AKT to its signalosome in the lipid raft. The
characteristics of OSU-T315 provide the following advantages. (1)
Shortcomings of recent strategies in developing targeted therapeutic
arise from the balancing between specificity and efficiency. As
multiple stimuli lead to AKT activation, ubiquitously inhibition of
this survival signal is challenging. The effect of OSU-T315 via AKT
regardless of upstream pathways activity may improve efficacy.
Contrary to current AKT inhibitors that abrogate phosphorylation to
selective isoforms, OSU-T315 prevents overall phosphorylation of
AKT by targeting raft translocation rather than showing selectivity
toward a particular isoform. (2) The functional redundancy of
different PI3Ks may result in the efficacy and drive drug resistance
for long-term treatment. Compared with existing PI3K inhibitors in
CLL clinical practice, OSU-T315 shows the potential to surpass the
issue. (3) The preferential cytotoxicity of OSU-T315 toward CLL
cells compared with normal B and T cells suggests a beneficial
therapeutic window.
PI3K/AKT signaling originates from lipid rafts following the
recruitment of AKT via the interaction of the PH domain of AKT
with PIP3 or PIP2.30,31 In B cells, Src family kinases including Lyn,
as well as the CD40 receptor, have been shown to be stably anchored
within lipid rafts. Certain proteins, including AKT, are accumulated
in rafts in part due to myristoylation. Our observations suggest that
OSU-T315 impairs the ability of AKT to be retained in cholesterol-
enriched lipid rafts as previously described,26 resembling the effect
of cholesterol inhibitors such as simvastatin.30,31 This effect was also
observed with synthetic antitumor lipids (ATLs). Consistent with the
assertions that both ATLs51,52 and cholesterol inhibitors30 exhibit
selective cytotoxicity toward malignant cells, a similar effect is

Table 7. Pharmacokinetic properties of OSU-T315

Discussion
Intravenous Orally Intraperitoneally
PK parameters (50 mg/kg) (50 mg/kg) (25 mg/kg)
The data report herein characterize a novel mechanism of action of
OSU-T315. Specifically, we demonstrate that OSU-T315 displaces Cmax (nM) 48 764 369.09 3604

AKT from lipid rafts, thus impairing AKT phosphorylation induced Tmax (minutes) 5 120 120
T1/2 (hours) 5.51 5.07 6.08
by a variety of pathways including BCR, CD40L, TLR9, and
Bioavailability (%) NA 5.8 58.6
integrin. The downstream consequences include downregulation
of Mcl-1 or Bcl-xl and caspase-dependent apoptosis. Remarkably, NA, not applicable.
BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 OSU-T315 ANTAGONIZES AKT LOCALIZATION IN RAFTS 291

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Figure 4. OSU-T315 impairs AKT translocation to lipid raft subdomains in plasma membrane. (A) Mec-1 cells were retro-virally transduced with pBABE-Myr-flag-AKT
vector (Addgene). The total lysates were subjected to western blot to verify the myristoylated AKT expression. (B) Mec-1 expressing Myr-flag-AKT was treated with increasing
concentration of OSU-T315. Total lysate was analyzed by immunoblotting. (C) Lipid raft from Mec-1 cells expressing Myr-flag-AKT was purified after vehicle or OSU-T315
(4 mM) treatment by the ultracentrifugation approach. The raft (R) and nonraft (NR) fractions were analyzed for raft-associated molecules, and Flotillin-1 serves as a lipid raft
marker. (D) The 3 independent studies were quantified for AKT content in raft compartment. (E) Mec-1 cells with Myr-flag-AKT were subject to immunofluorescence staining
with antibodies of a-AKT (Alexa 594) and a- Cholera toxin subunit B (CT-B) (Alexa 488). (F) The colocalization index was measured and analyzed by confocal microscope
double blindly. (G) 697 cells were treated with plate-bounded a-IgM, 4 mM OSU-T315, or in combination for 1 hour, and the lipid raft fractions were extracted for analysis.
Cholera toxin subunit B (CT-B) is the marker for lipid raft compartment.

observed in OSU-T315. These combined observations suggest that clinical activity in CLL. In contrast to ATLs that impact diverse
hypercholesterolemia might potentiate CLL development53 and also molecules in lipid rafts such as PI3K, PDK1, and mechanistic target
that our novel lipid raft-targeting pathway could show significant of rapamycin in addition to AKT,38 OSU-T315 appears to target
292 LIU et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

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Figure 5. OSU-T315 inhibits BCR-, CD40L-, and CpG-induced survival signal. (A) CLL cells were treated with either plate-bounded a-IgM, 0.5 mg/ml CD40L, 3.2 mM
CpG-ODN, or (C) plate-bound fibronectin or 1 mg/ml VCAM-1 combined with vehicle or 4 mM OSU-T315 for 15 minutes. The total lysate was subjected to immunoblotting.
(B,D) Data from individual patients were quantified for p-AKT level and normalized to GAPDH. (E-G) Cell viability was examined after 24 hours in the treated conditions of
plate-bounded a-IgM, 0.5 mg/ml CD40L, or 3.2 mM CpG-ODN combined with vehicle or different doses of OSU-T315.

AKT alone, indicating that this agent uniquely blocks the component
essential for AKT docking. We are currently investigating the under-
lying candidates by OSU-T315 using proteomic approaches.
OSU-T315 was originally designed to specifically disrupt the
interaction of AKT with its binding site on ILK, a putative PDK2
anticipated as a promising therapeutic target in several cancers.
Studies assessing the function of ILK in CLL or other leukemias are
lacking. Our data showed slightly lower amounts of ILK in CLL
compared with normal cells (supplemental Figure 2A-B), and ILK
knockdown could not recapitulate cytotoxicity by OSU-T315.
Together, these data do not support a strong role for ILK in CLL
progression. Besides being activated by phospholipase C-g2/protein
kinase C-b through the BCR pathway, ERK1/2 is also provoked via
diverse external signals, leading to CLL proliferation and survival.54
BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2 OSU-T315 ANTAGONIZES AKT LOCALIZATION IN RAFTS 293

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Figure 6. Prolonged survival of TCL1 leukemia-engrafted mice by OSU-T315. (A) C57BL/6 mice engrafted with TCL1 leukemia cells were treated orally with vehicle or 50 mg/kg
OSU-T315 daily after appearance of 10% leukemia cells in the peripheral blood. WBCs were monitored by blood smear slides weekly until euthanization was required. Data represent
WBC counts after 4-week treatment. (B) Overall survival was analyzed after treatment starts (n 5 6 per group). (C) C57BL/6 mice engrafted with TCL1 leukemia cells were treated by
intraperitoneal injection with vehicle or 25 mg/kg OSU-T315 once daily for 2 weeks after the appearance of 5% CD191, CD51 leukemia cells among CD451 peripheral blood
mononuclear cells. OSU-T315 was formulated in phosphate-buffered saline containing 10% Cremophor EL (Sigma). After a 2-week daily scheme, leukemic mice were treated every
other day with vehicle or 25 mg/kg OSU-T315 to prevent weight loss. The percent of leukemic cells was monitored weekly by flow cytometry until euthanization was required. Overall
survival was analyzed (n 5 9 for each group). (D) Data represent the pharmacokinetics of OSU-T315 in plasma after intravenous, intraperitoneal, or oral dosing (n 5 6 per group).

Interestingly, despite the comprehensive profiling by immunoblotting
showing reduction of tonic phospho-AKT and p-ERK on OSU-T315
treatment, only BCR- or CD49d-mediated ERK activation was con-
sistently abrogated by OSU-T315. Conversely, CD40L- or CpG-
induced signaling exhibited differential response in ERK status
(supplemental Figure 4), suggesting that OSU-T315 exclusively
suppressing PI3K/AKT signaling induced by diverse stimuli.
Finally, our in vivo study shows that, despite being hampered by
low oral bioavailability, OSU-T315 significantly prolonged survival
of leukemic mice. These promising data support the development of
improved formulation, delivery strategies, and/or derivatives, which
are being actively pursued by our group. Together, these findings pro-
vide an alternative strategy to target CLL cell survival by disrupt-
ing AKT recruitment to lipid rafts and introduce an outstanding
294 LIU et al BLOOD, 8 JANUARY 2015 x VOLUME 125, NUMBER 2

candidate for further investigation and development in CLL and
potentially other B-cell malignancies.
Acknowledgments
This work was supported by the Specialized Center of Research
from the Leukemia and Lymphoma Society, National Cancer
Institute grants P50-CA140158, R01 CA177292, R01 CA183444,
and P01 CA95426, the D. Warren Brown Foundation, the Four
Winds Foundation, The Sullivan Chronic Lymphocytic Leukemia
Research Fund, Mr and Mrs Michael Thomas, Al and Midge
Lipkin, and the Harry Mangurian Foundation.
Authorship
Y.L., J.A.W., K.B., and J.M. were involved in planning compo-
nents of the research, performed experiments, reviewed drafts, and
approved the final version of the paper; Y.-Y.Y. and E.H. were
involved in planning components of the research, reviewed drafts,
and approved the final version of the paper; X.Z. and A.L. performed
statistical analysis; S.B.S. and C.-S.C. were involved in planning
components of the research and provided necessary reagents es-
sential to the hypothesis of this paper; F.A., J.A.J., L.A.A., and
K.M. reviewed drafts and approved the final version of the paper;
and M.A.P., J.C.B., and A.J.J. planned every aspect of the pro-
posal, supervised the research, reviewed and modified drafts,
obtained funding for the research work, and approved the final
version of the paper.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: John C. Byrd, Rm 455B, 410 West 12th Ave,

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Columbus, OH 43210; e-mail: john.byrd@osumc.edu; or Amy J. Johnson,
Contribution: T.-M.L. designed the research, performed experi- Rm 455C, 410 West 12th Ave, Columbus, OH 43210; e-mail: amy.
ments, analyzed data, generated figures, and wrote the manuscript; johnson@osumc.edu.

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