Jeremy Grenier
PI3K inhibitors in CLL therapy
Dr. Panasci Lab
PI3K inhibitors in CLL therapy
Introduction
Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in Western
populations. It is a disease characterized by actively dividing B-lymphocytes in the lymph nodes
and bone marrow (Schmid et al. 1994; Messmer et al. 2005) as well as the accumulation of
quiescent lymphocytes in the peripheral blood of affected patients (Hamblin et al. 1997).
Overexpression of phosphatidylinositol 3-kinase (PI3K) in CLL B cells is thought to play a role
in this defect in apoptosis (Ringshausen et al. 2002). However, several classes of PI3K exist,
each with distinct downstream effects. This study focuses on class I PI3Ks which phosphorylate
inositol phospholipids, converting PtdIns(3,4)P2 to PtdIns(3,4,5)P3 (Hawkins et al. 1992). These
products act as docking sites for proteins containing a pleckstrin homology (PH) domain (Rameh
et al, 1997). One of the predominant results of this signaling is the recruitment of Akt through its
PH domain and its activation by PDK-1 (Astoul et al., 1999; Reif et al., 1997). This activation of
Akt is thought to contribute to CLL cell survival by blocking apoptotic pathways and promoting
proliferation and cell survival (Barragan et al., 2002; Ringshausen et al., 2002; Plate 2004).
Interestingly, the PI3K/Akt pathway is also implicated in chemoresistance in certain
cancers (Page et al., 2000; Brognard et al., 2001). For CLL patients, fludarabine (Flu) and
cyclophosphamide (HC) are standard treatments. Fludarabine acts as an adenine analogue,
incorporating into DNA during DNA synthesis. This incorporation inhibits DNA polymerase and
prevents cell cycle progression, often resulting in cytotoxicity (Gandhi et al. 1994).
Cyclophosphamide is metabolized in the liver to its activated form, 4hydroperoxycyclophosphamide. Cells in this study were treated with activated HC in place of
standard cyclophosphamide. This activated form acts as an alkylating agent to disrupt DNA
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Dr. Panasci Lab
replication, causing mutations leading to cell death (Teicher et al., 1996; Voelcker et al., 1984).
CLL patients often become resistant to these standard treatments, so methods of overcoming
resistance are an important area of CLL research. In one study, inhibition of PI3K increased
apoptosis in leukemic cells treated with fludarabine (Yu et al., 2005). Similarly, inhibition of
PI3K/mTOR with BEZ235, a dual PI3K/mTOR inhibitor, also sensitized T-ALL cells to
cyclophosphamide (Chiarini et al., 2010). These studies suggest that inhibition of class I PI3K
could offer clinical advantages when used with conventional chemotherapy.
Not surprisingly, inhibition of Akt induces apoptosis in CLL cells, foreshadowing the
clinical importance of the PI3K/Akt pathway in CLL (de Frias et al., 2009). As a single agent,
PI3K inhibitors have already been shown to decrease CLL lymphocyte survival. LY294002 is a
potent, class I PI3K inhibitor which induces apoptosis in CLL cells in vitro (Ringshausen et al.,
2002). However, LY294002 and other early developed PI3K inhibitors are relatively nonspecific
and show poor clinical application due to high toxicity levels (Schultz et al., 1995; Ng et al.,
2001; Knight et al., 2007). As a result, newer PI3K inhibitors are being developed. For example,
Herman et al. showed that a new class I PI3K inhibitor, CAL-101, promotes apoptosis in CLL
lymphocytes ex vivo (Herman et al., 2010).
The focus of this study is a novel PI3K inhibitor. NVP-BKM120 is a class I PI3K
inhibitor currently in clinical trials for solid tumors (Maira et al., 2010). BKM120 has already
been shown to act synergistically with Rapamycin, an mTOR inhibitor, in melanoma cell lines
(Aziz et al., 2010). However, it has yet to be studied in the treatment of CLL. In this study, we
explored the effects of BKM120 on CLL cell survival and chemoresistance.
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Dr. Panasci Lab
Materials and Methods
Cell Line Culture
I83, a B-CLL cell line, was derived from a patient with chronic lymphocytic leukemia
(Carlsson et al., 1989). MEC2, MEC1, JVM2, JVM3, JVM13, and EHEB were purchased from
DSMZ (Braunschweig, Germany). All cells were cultured in RPMI 1640 medium supplemented
with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) in a 5% CO2 humidified atmosphere.
Cytotoxicity Assay
B-CLL cell lines were plated on a 96-well plate in RPMI 1640 media/10% FBS at a
concentration of 1.5 × 105 cells/mL. After one hour, cells were treated with various
concentrations of NVP-BKM120 alone (Novartis, Cambridge, MA), fludarabine alone (Sigma
Aldrich Co, St. Louis, MO) , 4-hydroperoxycyclophosphamide alone (TCR, Toronto, Canada),
and combinations of fludarabine and BKM120 or 4-hydroperoxycyclophosphamide and
BKM120. Treated cells were incubated for 72 hours before treatment with the MTT or WST-1
assay as previously described by (Christodoulopoulos et al., 1998; Guertler et al., 2011). The
efficacies of the various drug treatments were determined by calculating 50% inhibitory
concentrations (IC50) and the synergy values (I value) according to the equation of Berenbaum
(Berenbaum, 1992). Using this equation I values significantly less than 1 indicate synergy, equal
to 1indicate additive behavior, and greater than 1 indicate inhibitory drug interactions.
Flow Cytometry Analysis
Cells were plated at 1.5 × 105 cells/mL in RPMI 1640 medium with 10% FBS. Cells were
treated with vehicle (DMF), BKM IC50, 0.1 µM BKM, 0.3 µM BKM, 0.5 µM BKM, Flu IC50,
HC IC50, and the combinations of primary drug with BKM. Cells were harvested 24 hours after
treatment. Apoptosis and phosphorylation of Akt were determined as described below.
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PI3K inhibitors in CLL therapy
Dr. Panasci Lab
pAkt Flow Cytometry
After harvesting, cells were washed in PBS before incubation in 1% paraformaldehyde at
37ºC for 10 min. Cells were then resuspended in ice cold 100% methanol. After washing in PBS3%FBS, samples were incubated at room temperature with anti-Akt S473-Alexa488 mouse
antibody (Invitrogen) for 30 min. A control sample was incubated with isotype-Alexa488
antibody (Invitrogen) for background measurement of fluorescence. All samples were then
washed and resuspended in PBS-3%FBS for analysis. Phosphorylation levels are expressed as a
percentage of the population.
Apoptosis Analysis
After harvesting, cells were washed in ice cold PBS and incubated in a solution of
binding buffer, propidium iodide (PI) , and Annexin V-APC for 15 min at room temperature
(Invitrogen). Control samples were incubated with binding buffer alone, binding buffer and PI,
or binding buffer and Annexin V-APC. Samples were then analyzed through flow cytometry.
Apoptosis is expressed as a percentage of the population.
Western Blot Analysis
3 x106 cells were plated at in RPMI media supplemented with 10% FBS and treated with
a range of non toxic concentrations of BKM120 (0-1 M) and the IC50 for 2, 6 and 24h to assess
the phosphorylation status of Akt. After treatment, cells were harvested and washed in PBS
before incubation in 100l of protein lysis buffer (20 mM Tris HCl, 150 mM NaCl, 1% NP40,
10% glycerol, 0.1% SDS, 1 mM Sodium orthovanadate, 1 mM PMSF and 1 protease/phosphatise
inhibitor complete mini pill (Roche Diagnostic Gmbh)) for 1h on ice. After a 15 minute
centrifugation at 13000 rpm, protein concentration was determined in the supernatant with the
protein BCA assay kit (Thermo Scientific). 50 g of protein was resolved in 4-15% SDS-PAGE,
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Dr. Panasci Lab
transferred to nitrocellulose (BioRad), and probed with the following antibodies: Akt and Akt
S473 (Cell signaling). Equal protein loading was confirmed by reprobing membranes with an
actin antibody (Santa Cruz). The blots were developed using the appropriate HRP-secondary
antibodies (α-mouse, α-rabbit or α-goat) and ECL (Amersham).
Results
BKM120 inhibits phosphorylation of Akt in a dose-dependent manner in CLL cell lines
Using Western blot analysis, we examined BKM120’s ability to inhibit PI3K by
assessing the phosphorylation status of its downstream target Akt. As shown in Figure 1, treating
cells with BKM120 reduces the phosphorylation of Akt in a dose-dependent manner, suggesting
that BKM120 is effective in inhibiting PI3K in MEC2 cells. From this, we chose relatively nontoxic concentrations of BKM120 which were just shown to inhibit activation of Akt. These
concentrations were used in the following combination experiment.
Fig. 1. Western blot with MEC2 cells 24 hours after treatment with BKM120
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Dr. Panasci Lab
BKM120 does not sensitize CLL cell lines to fludarabine or cyclophosphamide
Using the MTT and WST-1 assays, we determined the average IC50 values of BKM, Flu,
HC, and the combinations of Flu+BKM and HC+BKM in the I83 & MEC2 cell lines (Table 1).
Although there was a slight decrease in the average IC50 of Flu when combined with 0.1 µM
BKM in I83 cells, this difference does not suggest a synergistic response, as the average I-Value
remained near 1 (data not shown). These experiments were repeated in MEC2 cells with 0.2μM
& 0.1μM BKM. While there was a decrease in the IC50, these results are most likely additive
effects due to BKM’s toxicity at these concentrations (Table 2).
Cell Line
IC50
IC50 Flu
BKM (M) (M)
I83
1.07±0.43
0.93±0.19
MEC2
0.64±0.11
>20
IC50
F+0.2B
(M)
19±1.7
IC50
F+0.1B
(M)
IC50
4-HC
(M)
0.78±0.22
1.75±0.7
9
2.51±1.2
18.9±1.6
IC50
H+0.2B
(M)
IC50
H+0.1B
(M)
1.99±1.00
1.55±1.8
1.53±0.4
Table 1. Average IC50 Values determined through statistical analysis of MTT & WST-1 assay.
Cell Line
I-Value HC+
0.2M BKM
I83
MEC2
0.70±0.26
I-Value HC+
0.1M BKM
1.3±0.44
0.2M BKM % Survival
1.2±0.09
71.1±6.9
0.1M BKM % Survival
85.7±10.6
76.8±2.9
Table 2. Average I-Value and % Survival
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Dr. Panasci Lab
BKM120 only slightly reduces drug-induced phosphorylation of Akt
Fig. 2. Phosphorylation of Akt in I83 cell line. Flow cytometric analysis was used to assess
phosphorylation levels 24 hours after treatment.
80
% of cell population
70
60
50
40
30
20
10
0
Fig. 3. Levels of phosphorylated Ser473 Akt 24 hours after treatment, expressed as a percentage of cell
population.
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Dr. Panasci Lab
Fig. 4. Western blot analysis of phosphorylation status of Akt following 24hr treatment
Because the drugs were not acting synergistically, we sought to confirm that
phosphorylation of Akt was reduced when BKM120 was used in combination with the DNA
damaging drug Fludarabine. We used I83 for these experiments because MEC2 cells were not
sensitive to Flu alone. As shown in Fig. 2 and Fig. 3, phosphorylation of Akt dramatically
increases upon treatment with IC50 of Flu or HC, confirming results from earlier studies and
suggesting that this phosphorylation is upregulated after drug treatment. Treatment with varying
concentrations of BKM120 (0.5 µM, 0.3 µM, and 0.1 µM) shows only a slight reduction in
phospho-Akt levels, even at the toxic concentrations of 0.5M. We confirmed these observations
with a Western blot performed 24 hours after treatment. While concentrations above 0.6M
BKM show a clear reduction in pAkt levels, these concentrations are much too toxic to be useful
for combination studies. This increased phosphorylation of Akt may explain why BKM120 did
not sensitize cells to Flu or HC.
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Characterization of apoptotic signaling pathways
To confirm that the combination of drugs did not increase cell death, apoptotic signaling
protein levels were assessed 2 hours, 6 hours, and 24 hours after treatment in MEC2 cells. As
seen from the blots, all the apoptotic signaling molecules we assessed did not change over 24
hours regardless of treatment conditions. However, we did note a decrease in pAkt and pERK
levels as early as 2 hours post-treatment, confirming results from an earlier study using another
PI3K inhibitor (Yu et al. 2005). There appears to be a decrease in Bax protein in the 2 hour blot,
but this is most likely due to experimental error as the protein is restored after 6 hours and 24
hours. The same error most likely accounts for the reduced BCL-2 protein seen in the 24 hour
untreated control. Overall, these results suggest that the combinational treatment of Flu or HC
with BKM does not increase apoptotic signaling in these cells.
Fig. 5. Western blot analysis of apoptotic signalling proteins 2 and 6 hours post treatment.
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Dr. Panasci Lab
Fig. 6. Western blot analysis of apoptotic signalling proteins 24 hours post treatment.
Treatment with 0.1μM BKM120 does not increase Flu- or HC-induced apoptosis
We again sought to confirm earlier observations that the addition of a non-toxic
concentration of BKM120 does not increase Flu- or HC-induced apoptosis in I83 cells.
Preliminary data shows that Flu and HC induce apoptosis in a higher percentage of cells
compared to the vehicle DMF (Fig. 7 and Table 3). Consistent with the cytotoxicity data (Table
1), 0.1 µM BKM seems to have little effect on cellular apoptosis when used in combination with
Flu or HC. However, this observation must be confirmed with replicate experiments.
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Dr. Panasci Lab
Fig. 7. Induction of apoptosis in I83
cell line. Bottom right quadrant shows
early apoptotic cells. Top right
quadrant shows late apoptotic cells.
% Apoptotic Cells
DMF
0.1M
BKM
FLU IC50
FLU+0.1M
BKM
HC IC50
HC+0.1M
BKM
4.5
6.2
16.7
11.5
11.0
10.3
Table 3. Percentage of cells in early/late apoptosis as determined by flow cytometry.
Cytotoxicity of BKM120 alone on 7 CLL cell lines
While BKM120 may not sensitize CLL cells to Flu or HC, the IC50 values we obtained
for BKM120 alone in I83 and MEC2 were within clinically obtainable ranges. Therefore, we
next sought to determine if this high toxicity was consistent across other leukemic cell lines.
Results of WST-1 and MTT experiments are shown in Table 4 and Figure 8. Given the slightly
different IC50 values, we hypothesized that this discrepancy could be a result of different levels
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Dr. Panasci Lab
of basal pAkt expression between the cell lines. However, as seen in Figure 9, the basal phosphoSer473 levels do not correlate with sensitivity to BKM120. In the future, this experiment will be
repeated for phospho-Thr308Akt. This second phosphorylation site may better explain the
different toxicities to BKM. Lastly, we compared BKM120’s effectiveness in decreasing pAkt in
5 cell lines. As shown in Fig. 10, BKM120 reduces pAkt levels in all 5 cell lines except JVM3,
suggesting that BKM120 may inhibit growth in these cells by another mechanism.
Cell Line
I83
MEC2
MEC1
JVM2
JVM3
JVM13
EHEB
BKM120
IC50 (M)
1.07±0.43
0.64±0.11
0.84±0.04
0.78±0.18
1.18±0.29
1.34±0.75
0.68±0.15
Table 4. BKM120 IC-50 Values for 7 CLL cell lines.
2.5
BKM120 IC50
2
1.5
1
0.5
0
MEC2
EHEB
JVM2
MEC1
I83
JVM3
JVM13
Fig. 8. BKM120 IC50 Values
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Basal pAkt Ser473
Akt Ser473/Akt
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
JVM3
JVM13
MEC2
MEC1
JVM2
EHEB
I83
CLL Cell Lines
Fig. 9. Basal phospho-Ser473 Akt in 7 CLL cell lines as assessed by Western blot.
pAkt levels 24hr after treatment
1.6
Akt Ser473/Akt
1.4
1.2
MEC1 (n=1)
1
JVM3 (n=1)
0.8
JVM2 (n=1)
0.6
EHEB (n=1)
0.4
MEC2 (n=3)
0.2
0
Un
IC-50
0.2BKM
0.1BKM
0.05BKM
Fig. 10. Phosphorylation levels of Akt 24 hours after treatment with BKM120. Data
shown as ratio of pAkt/Akt.
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Discussion
BKM120 is a novel pan-class I PI3K inhibitor with improved clinical pharmacokinetics.
Given previous research done on the role of the PI3K/Akt pathway in CLL survival and
chemoresistance, we sought to sensitize CLL cells to standard chemotherapy by inhibiting class I
PI3K. However, our cytotoxicity results suggest that inhibition of PI3K with 0.1 or 0.2µM
BKM120 is not enough to sensitize CLL cell lines to chemotherapy, despite BKM120’s toxicity
at these concentrations.
While BKM120 does appear to reduce drug-induced Akt phosphorylation, it does not
reduce it to levels observed in the untreated control sample (Fig. 2). There are two possible
explanations for this: 1) Drug treatment causes a surge in PI3K activity which requires higher,
toxic doses of BKM120 to overcome, making the drug impractical for combination studies. 2) A
kinase other than PI3K is phosphorylating Akt in response to drug treatment. We have shown
that 0.2M BKM120 does reduce pAkt levels in cells not treated with chemotherapy. However,
as seen in Fig. 4 and Fig. 6, cells treated with both 0.2M BKM120 and a chemotherapeutic
agent still show significant levels of pAkt. This may be attributed to higher PI3K activity, but
this explanation seems unlikely without extracellular activation of PI3K.
For the second possibility, another kinase may be phosphorylating Akt in response to
chemotherapy. Several kinases have been discovered to phosphorylate Akt at S473 including
DNA-dependent protein kinase (Feng et al., 2004), Akt itself (Toker and Newton 2000), and
mTOR when bound to Rictor in the TORC2 complex (Santos et al., 2001; Sarbassov et al.,
2005). Thus, drug-induced phosphorylation of Akt may be attributed to the continued activity of
one or all of these kinases. This may account for the residual pAkt seen after cells were treated
with BKM120.
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Dr. Panasci Lab
If this is the case, these factors may also explain why BKM120 did not sensitize CLL
cells to fludarabine or cyclophosphamide. An alternative means of overcoming this resistance
may be inhibition of Akt directly rather than targeting PI3K upstream. Several Akt inhibitors
have been studied. For instance, Akti-1/2 and A-443654 effectively induce apoptosis in CLL Bcells ex vivo, providing another method of inhibiting the PI3K/Akt survival pathway (de Frias et
al., 2009). These inhibitors have yet to be studied in combination with conventional
chemotherapy in CLL cells.
As a single agent, BKM120 may yet prove useful. A study by Cuní et al. showed that
PI3K and NF-ĸB signaling are required to prevent spontaneous apoptosis of CLL cells ex vivo.
This PI3K signaling was dependent on cell culture with fibroblasts, suggesting that the
microenvironment promotes CLL cell survival through PI3K signaling (Cuní et al., 2004).
Because our study was performed in cell lines, this aspect of CLL survival was not addressed.
BKM120 may be more effective against CLL lymphocytes in vivo by disrupting the effects of
this microenvironment interaction pathway.
Future studies may also focus on combining BKM120 with an mTOR inhibitor to
overcome chemoresistance. This may further reduce the levels of pAkt seen after treatment with
BKM120 because, as explained above, mTORC2 is capable of phosphorylating and activating
Akt. Along these lines, a recent study shows that BKM120 and rapamycin, an mTOR inhibitor,
act synergistically to reduce cell viability in melanoma cell lines (Aziz et al., 2010). Similarly, a
dual PI3K/mTOR inhibitor, NVP-BEZ235, is cytotoxic to T-cell acute lymphoblastic leukemia
cells and sensitizes them to chemotherapy (Chiarini et al., 2010). Thus, dual inhibition of PI3K
and mTOR may also sensitize CLL lymphocytes to standard chemotherapy.
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Dr. Panasci Lab
In summary, BKM120 does not appear to sensitize CLL B-cells to fludarabine or
cyclophosphamide in vitro at the low doses used (0.1 µM & 2M) despite being very toxic as a
single agent (IC50 between 0.6µM-1.1 µM). This contradicts earlier studies showing that
inhibition of PI3K sensitizes leukemic cells to fludarabine (Yu et al., 2005). Our results may
reflect inefficiency in BKM120 activity when combined with other drugs or a difference in the
machinery responsible for chemoresistance in CLL cells (as opposed to other resistant
leukemias). Overall, these preliminary results predict inefficient sensitization of chemotherapy
with BKM120 in CLL cell lines. Despite this, further research of its effects on microenvironment
interactions are needed, as well as possible combination with a potent mTOR inhibitor.
Acknowledgments
I would like to thank Dr. Lawrence Panasci for giving me the opportunity to do this project, Dr.
Lilian Amrein for his support and technical assistance, and the McGill Department of Oncology
for funding my summer research.
References
1. Astoul E, Watton S, Cantrell DA. (1999). The dynamics of protein kinase B regulation during B
cell antigen receptor engagement. J. Cell Biol. 145, 1511-1520.
2. Aziz SA, Jilaveanu LB, Zito C, Camp RL, Rimm DL, Conrad P, Kluger HM. (2010). Vertical
Targeting of the Phosphatidylinositol-3 Kinase Pathway as a Strategy for Treating Melanoma. Clin
Cancer Res. 16:6029-39.
3. Barragán M, Bellosillo B, Campás C, Colomer D, Pons G, Gil J. (2002) Involvement of protein
kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic
leukemia cells. Blood. 99, 2969–2976.
4. Berenbaum M. Letter Correspondence re: “Greco et al., Application of a New Approach for the
Quantitation of Drug Synergism to the Combination of c/s-Diamminedichloroplatinum and l-tf-DArabinofuranosylcytosine. Cancer Res., 50: 5318- 5327, 1990.” Cancer Research. 1992 Aug; 52:
4558-4565.
5. Brognard J, Clark AS, Ni Y, Dennis PA. (2001). Akt/protein kinase B is constitutively active in
non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and
radiation, Cancer Res. 61: 3986–3997.
16
Jeremy Grenier
PI3K inhibitors in CLL therapy
Dr. Panasci Lab
6. Carlsson M, Totterman TH, Rosen A, Nilsson K. (1989). Interleukin-2 and a T cell hybridoma
(MP6) derived B cell-stimulatory factor act synergistically to induce proliferation and differentiation
of human B-chronic lymphocytic leukemia cells. Leukemia. 3: 593-601.
7. Chiarini F, Grimaldi C, Ricci F, Tazzari PL, Evangelisti C, Ognibene A, Battistelli M, Falcieri
E, Melchionda F, Pession A, Pagliaro P, McCubrey JA, Martelli AM. (2010). Activity of the
Novel Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235
against T-Cell Acute Lymphoblastic Leukemia. Cancer Res. 70: 8097-107.
8. Christodoulopoulos G, Muller C, Salles B, Kazmi R, Panasci L. (1998). Potentiation of
chlorambucil cytotoxicity in B-cell chronic lymphocytic leukemia by inhibition of DNA-dependent
protein kinase activity using wortmannin. Cancer Res 58:1789-1792.
9. Cuní S, Pérez-Aciego P, Pérez-Chacón G, Vargas JA, Sánchez A, Martín-Saavedra FM,
Ballester S, García-Marco J, Jordá J, Durántez A. (2004). A sustained activation of PI3K/NFkappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells. Leukemia. 18:
1391-400.
10. de Frias M, Iglesias-Serret D, Cosialls AM, Coll-Mulet L, Santidrián AF, González Gironès
DM, de la Banda E, Pons G, Gil J. (2009). Akt inhibitors induce apoptosis in chronic lymphocytic
leukemia cells. Haematologica 94:1698-1707.
11. Feng J, Park J, Cron P, Hess D, Hemmings BA. (2004). Identification of a PKB/Akt hydrophobic
motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem 279:41189–41196.
12. Gandhi V, Huang P, Plunkett W. (1994). Fludarabine inhibits DNA replication: a rationale for its
use in the treatment of acute leukemias, Leuk Lymphoma. 14 (Suppl. 2): 3–9.
13. Guertler A, Kraemer A, Roessler U, Hornhardt S, Kulka U, Moertl S, Friedl AA, Illig T,
Wichmann E, Gomolka M. (2011). The WST survival assay: an easy and reliable method to screen
radiation-sensitive individuals. Radiat Prot Dosimetry. 143: 487-490.
14. Hamblin TJ, Oscier DG. (1997). Chronic lymphocytic leukemia: the nature of the leukemic cell.
Blood Rev. 3: 119-28.
15. Hawkins PT, Jackson TR, Stephens LR. (1992). Platelet-derived growth factor stimulates synthesis
of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase. Nature 358, 157-159.
16. Herman SE, Gordon AL, Wagner AJ, Heerema NA, Zhao W, Flynn JM, Jones J, Andritsos L,
Puri KD, Lannutti BJ, Giese NA, Zhang X, Wei L, Byrd JC, Johnson AJ. (2010).
Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic
lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals. Blood. 116:
2078-88.
17. Knight ZA, Shokat KM. (2007). Chemically targeting the PI3K family. Biochem Soc Trans. 35(Pt
2):245-9
18. Maira M, Menezes D, Pecchi S, et al. Biological characterization of NVP-BKM120, a novel
inhibitor of phosphoinosotide 3-kinase in Phase I/II clinical trials. Presented at: 101st American
Association for Cancer Research Congress. April 17–21, 2010; Washington DC, US. Poster
19. Messmer BT, Messmer D, Allen SL, Kolitz JE, Kudalkar P, Cesar D, Murphy EJ, Koduru P,
Ferrarini M, Zupo S, Cutrona G, Damle RN, Wasil T, Rai KR, Hellerstein MK, Chiorazzi N.
(2005). In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic
leukemia B cells. J Clin Invest. 115:755-64.
20. Ng SS, Tsao MS, Nicklee T, Hedley DW. (2001). Wortmannin inhibits pkb/akt phosphorylation and
promotes gemcitabine antitumor activity in orthotopic human pancreatic cancer xenografts in
immunodeficient mice. Clin Cancer Res. 7(10):3269-75
21. Page C, Lin HJ, Jin Y, Castle VP, Nunez G, Huang M, Lin J. (2000). Overexpression of Akt/AKT
can modulate chemotherapy induced –apoptosis. Anticancer Res. 20: 407–416.
22. Plate JM. (2004) PI3-kinase regulates survival of chronic lymphocytic leukemia B-cells by
preventing caspase 8 activation. Leuk. Lymphoma. 45: 1519–1529.
23. Reif K, Burgering BMT, Cantrell DA. (1997). Phosphatidylinositol 3-kinase links the interleukin-2
receptor to protein kinase B and p70 S6 kinase. J. Biol. Chem. 272, 14426-14438.
17
Jeremy Grenier
PI3K inhibitors in CLL therapy
Dr. Panasci Lab
24. Rameh LE, Arvidsson AK, Carraway KL, Couvillon AD, Rathbun G, Crompton A,
VanRenterghem B, Czech MP, Ravichandran KS, Burakoff SJ, et al. (1997). A comparative
analysis of the phosphoinositide binding specificity of pleckstrin homology domains. J. Biol. Chem.
272, 22059-22066.
25. Ringshausen I, Schneller F, Bogner C, Hipp S, Duyster J, Peschel C, Decker T. (2002)
Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis
in B-CLL: association with protein kinase C. Blood. 100: 3741–3748.
26. Santos SC, Lacronique V, Bouchaert I, Monni R, Bernard O, Gisselbrecht S, Gouilleux F.
(2001). Constitutively active STAT5 variants induce growth and survival of hematopoietic cells
through a PI 3-kinase/Akt dependent pathway. Oncogene. 20:2080–2090.
27. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. (2005). Phosphorylation and regulation of
Akt/PKB by the rictor-mTOR complex. Science. 307:1098–1101.
28. Schmid C, Isaacson PG. (1994). Proliferation centres in B-cell malignant lymphoma,
lymphocytic (B-CLL): an immunophenotypic study. Histopathology. 24:445-51.
29. Schultz RM, Merriman RL, Andis SL. (1995). In vitro and in vivo antitumor activity of the
phosphatidylinositol-3-kinase inhibitor, wortmannin. Anticancer Res. 15(4):1135-9
30. Teicher BA, Chen YN, Ara G, Emi Y, Kakeji Y, Maehara Y, Keyes S, Northey D. (1996).
Interaction of interleukin-11 with cytotoxic therapies in vitro against CEM cells and in vivo against
EMT-6 murine mammary carcinoma. Int J Cancer. 67(6):864-70.
31. Toker A, Newton AC. (2000). Akt/protein kinase B is regulated by autophosphorylation at the
hypothetical PDK-2 site. J Biol Chem. 275:8271–8274.
32. Voelcker G, Wagner T, Wientzek C, Hohorst HJ. (1984). Pharmacokinetics of “activated”
cyclophosphamide and therapeutic efficacies. Cancer. 54: 1179-86.
33. Yu C, Mao X, Li WX. (2005). Inhibition of the PI3K pathway sensitizes fludarabine-induced
apoptosis in human leukemic cells through an inactivation of MAPK-dependent pathway. Biochem
Biophys Res Commun. 331(2):391-7.
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