Leukemia (2006) 20, 2093–2101
`& 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00
`
`www.nature.com/leu
`
`ORIGINAL ARTICLE
`
`Cytotoxic activity of gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia
`correlates with the expression of protein kinase Syk
`
`L Balaian and ED Ball
`
`Blood and Marrow Transplantation Division, Department of Medicine and Moores UCSD Cancer Center, University of California,
`San Diego, La Jolla, CA, USA
`
`Acute myeloid leukemia (AML) cells express the cell surface
`antigen CD33 that, upon ligation with a monoclonal antibody
`(mAb), is a downregulator of cell growth in a Syk-dependent
`manner. An anti-CD33 mAb coupled to a toxin, gemtuzumab
`ozogamicin (GO), is used for the treatment of AML (Mylotarg).
`Therefore, we investigated whether the response of AML cells
`to GO treatment also depends on Syk expression. Forty primary
`AML samples (25 Syk-positive and 15 Syk-negative) were tested
`for their response to the anti-proliferative effects of GO and
`unmodified anti-CD33 mAb. A correlation between Syk expres-
`sion and the response of leukemia cells to GO and anti-CD33
`mAb was found. ‘Blocking’ of Syk by small interfering RNA
`resulted in unresponsiveness of AML cells to both GO and anti-
`CD33 mAb-mediated cytotoxicity. Syk upregulation by the de-
`methylating agent 5-azacytidine (5-aza) induced re-expression
`of Syk in some cases, resulting in enhanced GO and anti-CD33-
`mediated inhibition of leukemia cell growth. Thus, the cytotoxi-
`city of both GO and anti-CD33 in primary AML samples was asso-
`ciated with Syk expression. 5-Aza restored Syk and increased
`the sensitivity of originally Syk-negative, non-responsive cells
`to CD33 ligation to levels of Syk-positive cells. These data
`have clinical significance for predicting response to GO and
`designing clinical trials.
`Leukemia (2006) 20, 2093–2101. doi:10.1038/sj.leu.2404437;
`published online 19 October 2006
`Keywords: myeloid leukemia; Syk; anti-CD33 mAb; gemtuzumab
`ozogamicin; 5-azacytidine.
`
`Introduction
`
`CD33 is a cell surface glycoprotein specifically expressed on
`myeloid cells including myeloid leukemia cells.1 Monoclonal
`antibodies (mAbs) against CD33 have been used in the diagnosis
`and therapy of acute myeloid leukemia (AML) for many years.2
`CD33, a member of the siglec family, is engaged in sialic acid-
`dependent cell interactions and adhesion of myeloid cells.3 The
`cytoplasmic tail of CD33 contains two immune tyrosine-based
`inhibitory motifs (ITIMs) and therefore, may serve as a potential
`inhibitory receptor.4–6 Engagement of CD33 induced apoptosis
`and inhibition of proliferation in leukemia cells from AML and
`chronic myeloid leukemia patients.7–10 However, little is known
`about the molecular mechanisms of the CD33 signaling events
`leading to inhibition of cell growth and apoptosis.
`The toxin (calicheamycin)-conjugated anti-CD33 mAb gem-
`tuzumab ozogamicin (GO)
`is now established as a useful
`
`Correspondence: Dr ED Ball, Blood and Marrow Transplantation
`Division, Department of Medicine and Moores UCSD Cancer Center,
`University of California, San Diego, 3855 Health Sciences Drive, La
`Jolla, CA 92093-0960, USA.
`E-mail: tball@ucsd.edu
`Received 25 April 2006; revised 11 August 2006; accepted 23 August
`2006; published online 19 October 2006
`
`component in the therapy of AML.11 GO induces remissions in
`about 30% of patients with relapsed AML. GO, a humanized
`immunoglobulin G (IgG) 4 mAb, contains human sequences
`whereas the complementarity-determining regions are derived
`from a murine antibody that binds CD33. The antibody is linked
`to N-acetyl-gamma calicheamycin via a bifunctional linker and
`is 50% loaded with 4–6 mol of calicheamycin per mole of
`antibody. Notably, the remaining 50% of the antibody is not
`linked to calicheamycin. Therefore, we examined whether the
`effects of GO depend on CD33-coupled molecules such as Syk.
`As GO is effective in only a minority of patients, the signaling
`activity of CD33 may be relevant to the responses achieved, in
`addition to the effect of the toxin calicheamycin.
`The protein kinase Syk is an essential element in many antigen
`receptor (B-cell receptor, T-cell receptor, Fc receptors) down-
`stream signaling cascades, resulting in cell responses such as
`adhesion, phagocytosis, proliferation and differentiation.12–17
`During early stages of antigen ligation, Syk binds to the recep-
`tor, becomes activated and phosphorylated and then phosphory-
`lates
`specific substrates
`such as phospholipase C-gamma
`with consecutive calcium influx.18 Late signaling events such
`as
`the activation of
`transcriptional
`factor nuclear
`factor-
`kappa B couple Syk activation with cell proliferation and
`differentiation.19
`in proximal signaling
`In myeloid cells, Syk involvement
`mediated by activated Fc receptor family members containing
`the tyrosine-based activation motif (ITAM) is well documen-
`ted.19–23 However, recent discoveries revealed that Syk is also
`involved in signaling of the ITIM-bearing CD22 receptor in B
`cells.24–27 We extended these findings by demonstrating that
`Syk (or ZAP-70) play an important role in CD33 signaling.28,29
`Upon CD33 ligation, Syk becomes phosphorylated and creates
`complexes with phosphorylated forms of the CD33 molecule
`itself and protein phosphatase Src homology phosphatase-1
`(SHP-1). Moreover, we showed that
`the anti-proliferative
`response of AML cells to CD33 ligation correlates with the
`level of Syk expression. Thirty percent of primary AML samples
`demonstrate no detectable Syk expression. Significantly greater
`numbers of Syk/Zap-70-positive samples respond to anti-CD33
`mAb treatment.29
`Recent discoveries established Syk as a tumor suppressor and
`linked deficient Syk expression to a variety of human
`hematopoietic30–33 and solid tumors.33–35 In breast cancer,
`Syk kinase is a potent modulator of malignant growth and a
`potential
`tumor suppressor, presumably by controlling cell
`division.33 Moreover,
`it was demonstrated that
`loss of Syk
`expression in breast cancer and T-cell acute lymphoblastic
`leukemia cells occurs at the transcriptional level, and is a result
`of DNA hypermethylation.31,36,37 Treatment of these cells with
`5-azacytidine (5-aza), a methylation inhibitor, restored Syk
`expression and function.36,37
`
`1 of 9
`
`BI Exhibit 1019
`
`

`

`2094
`
`Cytotoxicity of anti-CD33 mAb and GO correlate with Syk expression
`L Balaian and ED Ball
`
`5-Aza is a nonspecific Syk DNA methyltransferase inhibitor
`that is used for the treatment of myelodysplasia38 and possibly
`AML.39,40 However, the effects of 5-aza on leukemia cell growth
`are not well defined. As expression levels of Syk are likely to
`play an important role for the competency of the immune system
`and may also play a role in oncogenesis, we examined a panel
`of 40 primary AML samples for possible correlations between
`Syk expression and their response to the anti-proliferative effects
`of unmodified or immunotoxin-bound anti-CD33 mAb.
`
`Materials and methods
`
`Antibodies
`The anti-CD33 mAb was obtained from Medarex Inc. (Prince-
`ton, NJ, USA). Anti-Syk, anti-SHP-1, anti-CD33 rabbit or goat
`polyclonal antibodies were purchased from Santa Cruz Bio-
`technology Inc. (Santa Cruz, CA, USA). The anti-CD13 mAb was
`purchased from Cell Sciences Inc. (Norwood, MA, USA). A horse-
`radish peroxidase-conjugated anti-phosphotyrosine mAb, 4G10,
`was obtained from Upstate Biotechnology Inc. (Lake Placid,
`NY, USA).
`
`Materials
`All chemicals were purchased from Sigma Chemical Co. (St
`Louis, MO, USA).
`
`Cells
`The human AML cell line Hodgkin’s lymphoma (HL)-60 was
`obtained from ATCC (Manassas, VA, USA). Cells from AML
`patients were collected from peripheral blood after informed
`consent under the auspices of the University of California San
`Diego Institutional Review Board. Mononuclear cells were
`isolated on a Ficoll-Hypaque gradient and cultured under
`standard conditions
`in Rosewell Park Memorial
`Institute
`medium (RPMI)-1640 containing 10% fetal calf serum (FCS)
`and 100 ng/ml of granulocyte-monocyte colony-stimulating
`factor.
`
`Proliferation assay
`AML cells were cultured in triplicate wells in 96-well round
`bottom plates, with or without various amounts of anti-CD33 or
`control anti-CD13 mAb in a final volume of 200 ml of RPMI-10
`at 371C in humidified 5% CO2 in air, at 105 cells per well. The
`cultures were pulsed with 1 microCi [3H]-thymidine per well
`during the last 3 h of the 48 h culture for cell lines. Primary AML
`cells were pulsed during the last 16 h of culture. The amount of
`3H-thymidine incorporated into acid-precipitable DNA was
`assessed via liquid scintillation counting.
`
`Flow cytometry analysis
`Leukemia cells were washed and then suspended in staining
`media (SM), containing RPMI-1640, 3% FCS, 0.01% NaN3 and
`1 mg/ml propidium iodide (Calbiochem, La Jolla, CA, USA), plus
`saturating amounts of fluorescein isothiocyanate-conjugated
`anti-CD33, anti-CD64 mAbs, or an isotype-matched control
`mAb of irrelevant specificity. After 30 min at 41C, the cells were
`washed in SM twice and then analyzed on a FACScan (Becton
`Dickinson, San Jose, CA, USA). Dead cells and debris were
`excluded from analysis by characteristic forward and side-
`scatter profiles and propidium iodide staining. The immuno-
`phenotype of primary AML peripheral blood mononuclear cells
`
`Leukemia
`
`was determined by flow cytometry. CD2, CD3, CD7, CD10,
`CD13, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD64,
`CD95, human leukocyte antigen-DR, TdT expression were
`analyzed. All antibodies were obtained from BD Pharmingen
`(San Jose, CA, USA).
`
`Cell transfection
`SMARTpool small interfering RNAs (siRNAs) (collection of at
`least four individual siRNA, catalog No. 60–047 ) that target
`human Syk (Gen Bank Accession No. NM_003177) as well as
`control RNA (nonspecific pool) were purchased at Upstate
`Biotechnology Inc. (Lake Placid, NY, USA). Leukemia cells were
`transfected according to the manufacturer’s instructions. Briefly,
`cells (3–4 106) were transfected by 5 mg of Syk siRNA or
`control naked siRNA mix using 6 ml Lipofectamine 2000
`(Invitrogen, Carlsbad, CA, USA). Seventy-two hours after
`transfection, the cells were used for proliferation assays and
`Western blot analysis.
`
`Cell activation and immunoprecipitation
`Mononuclear cells from AML patients or AML cell lines (5–
`6 106) were activated by 10 mg/ml anti-CD33 or anti-CD13
`mAb of similar isotype for 20 min at room temperature, followed
`by addition of polyclonal anti-mouse IgG at 20 mg/ml
`for
`variable times (1–60 min). The reaction was stopped by adding
`ice-cold phosphate-buffered saline (PBS). After three washes in
`ice-cold PBS, the cells were lysed in lysis buffer, containing 1%
`(v/v) Triton X-100, 0.15 M NaCl, 50 mM Tris-HCl (pH¼ 7.2),
`0,1% sodium dodecyl sulfate (SDS), 1 mM Na-orthovanadate,
`1 mM phenylmethylsulfonyl fluoride, 1% (v/v) ethylenediamine-
`tetraacetic acid, 1% (v/v) Aprotinin and 0.03 mM Leupeptin.
`After 30 min on ice,
`the nuclear debris was removed by
`centrifugation for 15 min at 13 000 g. Lysates were equalized
`with respect to the amount of protein as assessed by optical
`density at 280 nM. Specific immunoprecipitation was performed
`for 2 h to overnight in the presence of 30% (vol./vol.) ‘Protein
`A/G’, conjugated with agarose (Santa Cruz Biotech., Santa Cruz,
`CA, USA). Immunoprecipitates were washed three times in lysis
`buffer and then suspended in equal volume of Laemli sample
`buffer for SDS-polyacrylamide gel electrophoresis (PAGE).
`
`SDS-PAGE and Western Blotting
`Total cell lysates or immunoprecipitates were added to separate
`wells (8 mg/well) of SDS-PAGE (7.5–10% acrylamide) gel,
`electrophoretically size-separated under reducing conditions,
`and then transferred onto nitrocellulose for immunoblotting. The
`filters were first incubated for 1 h in 5% non-fat dry milk in PBS-
`T (PBS plus 0.01% Tween 20), and then incubated with the
`primary antibody for 2 h. After washing in PBS-T, the filters were
`incubated for 1 h in horseradish peroxidase-conjugated match-
`ing secondary antibodies. The filters were then washed in PBS-T,
`incubated with the enhanced chemoluminescence detection
`reagents (Pierce, Rockford, IL, USA), and exposed to X-ray film
`(Fuji Film, Fischer, Tustin, CA, USA). In all figures, samples were
`run in parallel gels.
`
`Statistical analyses
`S.e.m. calculation, graph production and statistical evaluation
`were performed using Sigma-Plot 8.0 version (Systat Software
`Inc., Point Richmond, CA, USA) and Microsoft Excel (Microsoft,
`Seattle, WA, USA). Statistical significance of
`the difference
`between sample groups was calculated by using the Student’s
`
`2 of 9
`
`BI Exhibit 1019
`
`

`

`t-test and was defined as a P-value less than or equal to 0.05.
`The significance of observed differences in proportions was
`tested using the w2 test and was defined as a Pp0.05.
`
`Results
`
`The growth inhibitory effects of anti-CD33 mAb:
`correlation with Syk expression
`The cell surface CD33 receptor on myeloid leukemia cells
`functions as a negative regulator of cell growth. Anti-CD33
`mAb-mediated growth arrest occurs
`in a dose-dependent
`
`Table 1
`
`Syk expression on primary AML samples
`
`FAB
`
`MO
`M1
`M2
`M3
`M4
`M5
`M6
`M7
`Unknown
`
`Total no.
`of
`samples
`
`CD33
`expression
`(median) (%)
`
`No. of Syk-
`positive
`samples
`
`No. of Syk-
`negative
`samples
`
`1
`5
`7
`1
`10
`9
`2
`1
`4
`
`92
`81
`85
`73
`86
`94
`85
`92
`83
`
`1
`2
`5
`1
`5
`5
`2
`1
`3
`
`0
`3
`2
`0
`5
`4
`0
`0
`1
`
`Abbreviations: AML, acute myeloid leukemia; FAB, French-American-
`British.
`
`2095
`
`Cytotoxicity of anti-CD33 mAb and GO correlate with Syk expression
`L Balaian and ED Ball
`
`than 0.1 mg/ml.28,29 At
`manner, at concentrations greater
`optimal concentrations (0.1 mg/ml), anti-CD33 mAb inhibited
`colony formation of HL-60 cells by 450% and DNA synthesis
`in primary AML cells up to 40%. We previously found that
`CD33 ligation induced tyrosine phosphorylation of the tyrosine
`kinase Syk, but not of src-family tyrosine kinases (Fyn, Lyn).28,29
`As these results suggested an important role of Syk in CD33
`signaling, we tested a panel of 40 primary AML samples from
`different French-American-British (FAB) types for Syk expression
`(Table 1). All samples had high blast counts (data not shown),
`and the majority (36 of 40) contained high percentages (460%)
`of surface CD33. By Western blotting (data not shown), Syk
`expression was undetectable in about 37% (15 of 40) of primary
`AML samples. We did not detect any correlation between Syk
`expression and FAB type or CD33 expression.
`We previously reported that anti-CD33 mAb induced dose-
`dependent growth inhibition more effectively in primary AML
`cells that expressed Syk.29 Here,
`in order
`to amplify the
`representative groups, a panel of 40 primary AML samples
`was tested for their response to the optimal concentration
`(0.1 mg/ml) of anti-CD33 mAb (Figure 1a). The level of inhibition
`in Syk-positive samples was considerably higher (mean value
`435%) than in Syk-negative samples (mean value o20%). This
`difference was statistically significant (Po0.05; Student’s t-test).
`Moreover, among Syk-positive samples (n¼ 25), 68% demon-
`strated significant growth inhibition in response to CD33
`ligation (responders, as defined by 425% inhibition of 3H-
`thymidine uptake by anti-CD33 mAb) whereas 32% had no
`significant response (non-responders, defined by o25% growth
`inhibition) (Figure 1b). In contrast, only 20% of the Syk-negative
`
`a-CD13 mAb
`a-CD33 mAb
`
`( n = 2 5 )
`
`S y k +
`
`( n = 1 5 )
`
`S y k -
`
`
`
`Syk - samples
`(15 of 40)
`
`R2
`
`0%
`
`non-R
`32%
`
`P =0.0033
`Chi-square test
`
`non-R
`80%
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`(% of control media basal level)
`
`Inhibition of proliferation
`
`Syk + samples
`(25 of 40)
`
`a
`
`b
`
`R6
`
`8%
`
`Figure 1 Correlation between Syk expression and the anti-proliferative effect of CD33 ligation in primary AML cells. (a) Proliferation assay.
`Primary AML cells were cultured for 48 h in the presence or absence of 0.1 mg/ml of anti-CD33 mAb or control anti-CD13 mAb. 3H-thymidine
`incorporation was measured during the last 18 h of culture. Basal proliferation in the absence of antibody (c.p.m.45500 c.p.m.) was considered 0,
`and results are expressed as % change for each condition. Samples were grouped according to the expression of Syk/Zap70 and their response to
`CD33 ligation. Responders (R) are defined as samples with 425% inhibition of 3H-thymidine uptake in the presence of anti-CD33 mAb, whereas
`non-responders (NR) are defined as samples with o25% inhibition. Error bars indicate the s.e.m. (b) Correlation between Syk expression and the
`response of AML primary cells to CD33 ligation. Summary of primary AML proliferation.
`
`Leukemia
`
`3 of 9
`
`BI Exhibit 1019
`
`

`

`Cytotoxicity of anti-CD33 mAb and GO correlate with Syk expression
`L Balaian and ED Ball
`
`2096
`
`samples (n¼ 15) responded to anti-CD33 mAb. These results
`suggest a correlation between Syk expression and responsive-
`ness of AML cells to CD33 ligation (P¼ 0.0033 by w2 test).
`
`The growth inhibitory effects of GO (immunotoxin-
`conjugated anti-CD33): correlation with Syk expression
`þ
`As Syk
`AML cells were more likely to respond to CD33
`
`ligation with growth inhibition compared to Syk
`AML samples,
`we asked whether the anti-proliferative effect of GO might also
`depend on the presence of Syk. GO inhibited DNA synthesis in
`a dose-dependent manner in both Syk-positive and Syk-negative
`samples, but the level of inhibition was significantly higher in
`þ
`compared to Syk samples at all concentrations tested
`the Syk
`(Po0.003, Student’s t-test) (Figure 2a). The difference was more
`prominent at low doses of GO and diminished at higher doses,
`probably owing to free toxin activity.
`Response to GO was defined as 450% inhibition of 3H-
`thymidine uptake at 10 ng/ml, whereas non-responders demon-
`strated o50% inhibition at this concentration. In Syk-positive
`AML samples, the number of responsive samples was signifi-
`cantly higher (73%) compared to Syk-negative samples (17%)
`(Figure 2b). These data show a correlation between Syk
`expression in primary AML cells and the inhibitory activity of
`GO (P¼ 0.02; w2 test).
`
`siRNA-mediated silencing of Syk expression largely
`prevents the anti-proliferative response of AML cells
`to anti-CD33 mAb and GO
`To determine whether Syk expression was required for CD33-
`mediated inhibition of DNA synthesis, we used a siRNA strategy
`to downregulate Syk expression. We transfected HL-60 cells
`with Syk-specific siRNA or control siRNA using Lipofectamine
`2000 (Figure 3). Seventy-two hours post-transfection, Syk was
`undetectable by Western blotting in cells treated with the Syk-
`siRNA,
`the level of SHP-1 was unchanged in these cells,
`demonstrating specificity of the effect (Figure 3c). Downregula-
`
`tion of Syk expression largely prevented the anti-proliferative
`effects of the naked anti-CD33 mAb (Figure 3a) as well as the
`immunotoxin-conjugated mAb GO (Figure 3b). Thus,
`the
`Syk-specific
`siRNA converted initially
`Syk-positive
`and
`CD33-responsive AML cells into Syk-negative non-responsive
`cells. These results show that Syk plays a central role in CD33
`signaling.
`
`5-Aza treatment of Syk-negative primary AML cells
`increases their response to GO and anti-CD33 mAb
`Syk is an important tyrosine kinase, which appears to function as
`a tumor suppressor that is silenced by hypermethylation in many
`cancer cells. As we found a correlation between Syk expression
`and the response of primary AML samples to GO and anti-
`CD33 m Ab, the DNA methylase inhibitor 5-aza was used in a
`panel of 40 primary AML samples alone (Figure 4) and in
`combination with GO or anti-CD33 mAb (Figure 5).
`First, we analyzed dose- and time-dependent anti-prolifera-
`tive effects of 5-aza on human leukemia cell
`line HL-60
`(Figure 4a). Treatment conditions: dose of 100 nM for 48 h was
`considered as
`suboptimal, and was used in consequent
`experiments with primary AML cells. Based on the level of
`inhibition mediated by 5-aza, we distinguished three groups of
`primary AML samples (Figure 4b). The majority (21 of 40
`samples) demonstrated low response (inhibition of proliferation
`o25%).
`In nine of 40 samples, 5-aza treatment
`induced
`medium (25–50% inhibition) and in 10 of 40 samples this treat-
`ment mediated high (450% inhibition) responses. Response to
`5-aza was defined as 425% inhibition of 3H-thymidine uptake
`at 100 nM after 48 h treatment, whereas non-responders
`demonstrated o25% inhibition at these treatment conditions.
`In Syk-positive AML samples, the number of responsive samples
`was
`significantly higher
`(60%) compared to Syk-negative
`samples (26%) (Figure 4c). These data suggest a correlation
`between Syk expression in primary AML cells and the inhibitory
`activity of 5-aza (P¼ 0.04; w2 test).
`
` Syk + (n =25) AML samples
` Syk - (n=15) AML samples
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
` Proliferation (% of control)
`
`a
`
`b n
`
`on-R
`36%
`
`none
`
`1
`
`
`
`5
`10
`50
`100
`GO concentration (ng/ml)
`
`500
`
`1000
`
`Syk + samples (25 of 40)
`
`Syk- samples (15 of 40)
`
`R1
`
`7%
`
`P = 0.02;
`Chi-square test
`
`non-R
`83%
`
`R6
`
`4%
`
`Figure 2 The effect of GO on growth of AML cells is dependent on Syk expression. (a) Dose-dependent inhibition of proliferation induced by GO
`treatment. Proliferation assays were performed with primary AML cells as described in Figure 1a. GO was added for 48 h at the indicated
`concentration. Basal cell proliferation of untreated cells (410 000 c.p.m. of 3H-thymidine incorporation) was considered to be 100%.
`(b) Correlation between Syk expression and the response of primary AML cells to GO treatment. Summarized data (mean) for 25 Syk-positive and
`15 Syk-negative samples are presented. Error bars indicate the s.e.m.
`
`Leukemia
`
`4 of 9
`
`BI Exhibit 1019
`
`

`

`Cytotoxicity of anti-CD33 mAb and GO correlate with Syk expression
`L Balaian and ED Ball
`
`2097
`
`1
`10
`100
`none
`1000
`GO concentration (ng/ml)
`untreated cells
`control si-RNA treated
`Syk si-RNA treated
`
`120
`100
`80
`60
`40
`20
`0
`
`b
`
`Proliferation (% of control)
`
`*
`
`*
`
`*
`
`cntrl RNA Syk si-RNA
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`a
`
`Proliferation (% of control)
`
`media
`α-CD13 mAb
`α-CD33 mAb
`
`c
`
`I
`
`IP
`
`IgG
`1 2 3 4
`
`Syk
`1 2 3 4
`
`I I
`
`IP
`
`IgG
`1 2 3 4
`
`SHP-1
`1 2 3 4
`
`1 – untreated cells
`2 – control RNA treated cells
`3 – Syk siRNA treated cells
`4 – positive control (I-Raji or II-U-937 cells)
`
`probed with
`α-Syk Ab
`Syk
`
`probed with
`α-SHP-1 Ab
`SHP-1
`
`Figure 3 siRNA silencing of Syk expression decreases the anti-proliferative response of primary AML cells to anti-CD33 mAb including GO.
`(a and b) Proliferation assay. HL-60 cells were transfected with Syk siRNA or control siRNA. Seventy-two hours post-transfection, cells were treated
`with either naked anti-CD33 mAb or control anti-CD13 mAb (a) or they received the indicated concentrations of GO (b). Proliferation assays were
`performed as described in Figure 1a. The basal 3H-thymidine incorporation of untreated cells was 410 000 c.p.m. (considered as 100%). Data
`represent the mean7s.e.m. of three independent experiments. The asterisks indicate statistically significant difference between groups of data.
`(c) Western blots of HL-60 cells transfected with Syk siRNA or control siRNA. After 72 h of culture, cell
`lysates were subjected to
`immunoprecipitation and Western blotting with the indicated mAb. Shown results represent one of three independent experiments.
`
`2
`
`=
`
`w ( n
`
`l o
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`b
`
` (% of untreated cell proliferation)
`
`Inhibition of proliferation
`
`9 )
`
`=
`
`d i u m ( n
`
`0 )
`
`1
`
`=
`
`h ( n
`
`1 )
`h i g
`m e
`Group response to 5-aza treatment
`
`Syk+ (n=15) samples
`n-R
`n=11,
`73%
`
`R
`n=4,
`27%
`
`R
`n=15,
`60%
`
`P=0.04
`Chi-square test
`
`5 aza 100 nM
`5 aza 1000 nM
`
`none
`
`1
`2
`4
`Days in culture
`
`5
`
`c
`
`Syk+ (n=25) samples
`
`n-R
`n=10,
`40%
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`a
`
`Proliferation (% of control)
`
`Figure 4 The effect of 5-aza on growth of AML cells is dependent on Syk expression. (a) Proliferation assay. HL-60 cells were treated with
`indicated concentrations of 5-aza for 1–5 days. After two washes, proliferation assays were performed as described in Figure 1a. The basal
`3H-thymidine incorporation of untreated cells was 410 000 c.p.m. (considered as 100%). Data represent the mean7s.e.m. of three independent
`experiments. (b) Proliferation assay. Primary AML cells were cultured for 48 h in the presence of 100 nM of 5-aza and then after two washes,
`proliferation assays were performed as described in Figure 1b. (c) Correlation between Syk expression and the response of primary AML cells to
`5-aza treatment. Summary of primary AML cell proliferation.
`
`Leukemia
`
`5 of 9
`
`BI Exhibit 1019
`
`

`

`Cytotoxicity of anti-CD33 mAb and GO correlate with Syk expression
`L Balaian and ED Ball
`
`2098
`
`Syk+ samples (n=25)
`
`Syk- samples (n=15)
`
`b
`120
`100
`80
`60
`40
`20
`0
`none
`1
`α-CD33 mAb concentration (μg/ml)
`
`none
`
`0.1
`
`0.1
`
`1
`
`Syk- samples (n=15)
`
`Syk+ samples (n=25)
`
`d
`120
`100
`80
`60
`40
`20
`0
`
`none
`
`1
`
`10
`
`100
`GO concentration (ng/ml)
`
`none
`
`1
`
`10
`
`100
`
`a
`120
`100
`80
`60
`40
`20
`0
`
`c
`120
`100
`80
`60
`40
`20
`0
`
`Proliferation (% of conrol)
`
`Figure 5 5-Aza treatment of primary AML cells increases response to anti-CD33 mAb and GO. Primary AML cells were pretreated with 5-aza
`(100 nM) for 48 h. After two washes, proliferation assays were performed as described in Figure 1a. Summarized data (mean) for 25 Syk-positive
`(a and c) and 15 Syk-negative (b and d) samples are presented. Error bars indicate the s.e.m.
`
`no treatment
`5-aza (100nM ) 48h treatment
`
`Next, we analyzed the effects of the combined treatment of
`AML primary cells with 5-aza and then with either anti-CD33
`mAb or GO (Figure 5). In Syk-positive samples, pretreatment
`with 5-aza enhanced (about 20%) the anti-proliferative effects
`of antibody (Figure 5a) or GO (Figure 5c). Similar experiments
`with control isotype-matching anti-CD13 mAb did not result
`in increased growth inhibition (data not shown). Meanwhile,
`in Syk-negative primary samples, pretreatment with 5-aza led to
`statistically significant (Po0.05, Student’s t-test) enhancement
`of growth inhibition (up to 40% increase) induced by GO or
`anti-CD33 mAb (Figure 5b and d).
`We examined whether treatment with 5-aza may restore Syk
`expression in Syk-negative AML cells through induction of
`generalized DNA hypomethylation. In 15 Syk-negative primary
`AML samples, five (33%) showed restored Syk expression after
`5-aza treatment (Figure 6a and b). In Syk-positive samples, the
`level of Syk expression was unchanged. Of note, in two of five
`(40%) SHP-1-negative primary AML samples, 5-aza treatment
`also restored SHP-1 expression (data not shown).
`Treating Syk-positive AML cells with single-agent 5-aza at
`100 nM inhibited DNA synthesis by almost 40%, whereas the
`same drug concentration caused o20% inhibition in Syk-
`negative cells (Figure 6c). 5-Aza in combination with GO or
`anti-CD33 caused less than additive inhibition of DNA synthesis
`in Syk-positive cells (Figure 6c, left panel).
`induce re-
`In Syk-negative cells where 5-aza did not
`expression of Syk,
`the addition of GO or anti-CD33 mAb
`enhanced the anti-proliferative effect of 5-aza (Figure 6c, middle
`panel).
`In contrast, Syk-negative AML cells, where 5-aza
`induced re-expression of Syk, were only marginally inhibited
`by 5-aza or anti-CD33 mAb alone, whereas the combination
`produced a more than additive effect in AML cells (Figure 6c,
`right panel). Even the effect of GO was almost doubled by 5-aza
`in these cells. Thus, total inhibition of DNA synthesis in the
`
`presence of 5-aza plus GO or anti-CD33 mAb reached 60–70%
`and was similar in Syk-positive and Syk-negative cells.
`
`Discussion
`
`the anti-proliferative
`time that
`We demonstrate for the first
`effects of GO in AML is associated with the expression of Syk
`and that 5-aza treatment of Syk-negative and, therefore, poorly
`responsive, AML cells restored sensitivity to CD33 ligation-
`induced cytotoxicity mediated by both GO and unconjugated
`anti-CD33 mAb.
`ligation of CD33 molecules
`Recent studies showed that
`mediated AML cell growth inhibition,7–10 activation of protein
`tyrosine phosphatases SHP-1 and SHP-2,3–5 and a recruitment of
`the Syk protein tyrosine kinase.28,29 These events led to
`apoptosis and inhibition of cell growth. Our previous experi-
`ments also proved that the inhibitory effect of anti-CD33 mAb
`was caused by ligation of the CD33 molecule itself rather than
`through Fc receptor crosslinking.41 We also demonstrated that
`Syk expression was associated with the response of leukemia
`cells to the anti-proliferative effect of unconjugated anti-CD33
`mAb.29 In this study, we expanded these observations in a larger
`panel of primary AML samples (n¼ 40). Western blots detected
`25 (63%) Syk-positive and 15 (37%) Syk-negative samples.
`These data confirm our previous findings
`that Syk was
`undetectable in about 30% primary AML samples and that the
`expression of Syk correlated with response to CD33 ligation.21
`Significant differences in CD33 signaling between responsive
`and non-responsive AML cells that expressed Syk were found
`(29 and data not shown). In CD33-responsive AML samples,
`CD33 ligation induced tyrosine phosphorylation of Syk and
`Syk/CD33 and Syk/SHP-1 de novo complex formation. How-
`ever, CD33 signaling in CD33-non-responsive AML samples
`
`Leukemia
`
`6 of 9
`
`BI Exhibit 1019
`
`

`

`Cytotoxicity of anti-CD33 mAb and GO correlate with Syk expression
`L Balaian and ED Ball
`
`2099
`
`a
`
`b
`
`Syk-/-,
`n=10, 25%
`
`t
`n
`a (
`
`t m e
`z
`a
`-
`
`a
`
`5
`
`0
`
`0
`
`1
`
`e
`
`o t r
`
`n
`
`)
`
`h
`
`8
`
`n M 4
`
`Syk+,
`n=25, 62%
`
`Syk-/+,
`n=5, 13%
`
`AML #13 – (1 of 25 Syk+)
`
`AML #9 – (1 of 5 Syk-/restored)
`
`AML #11 – (1 of 10 Syk-/non-restored)
`
`80
`
`60
`
`40
`
`20
`
`0
`
`II.Syk -/- (n=10;25% of total)
`*
`
`80
`
`*
`
`none a-CD13 a-CD33 GO
`no treatment
`5-aza (100 nM 48 h)
`
`60
`
`40
`
`20
`
`0
`
`III.Syk -/+(n=5; 13% of total)
`
`*
`
`*
`
`none a-CD13 a-CD33 GO
`
`I.Syk + (n=25;63% of total)
`
`none a-CD13 a-CD33 GO
`
`80
`
`60
`
`40
`
`20
`
`0
`
`(% of control)
`
`c
`
`Inhibition of proliferation
`
`Figure 6 5-Aza treatment of Syk-negative primary AML cells increases response to anti-CD33 mAb and GO. (a and b) Western blot data. At 48 h,
`cells were harvested and analyzed by Western blotting for Syk expression. Syk/ is an abbreviation for Syk-negative samples where Syk was not
`restored by 5-aza. Syk/þ is an abbreviation for Syk-negative samples where Syk was restored by 5-aza treatment. (c) Proliferation assay. Primary
`AML cells were treated with 5-aza for 48 h, washed and cultured in the presence or absence of anti-CD33 or CD13 mAb (0.1 mg/ml) or GO
`(0.01 mg/ml) for 48 h and the proliferation assay performed as described in Figure 1. Bars marked with asterisk have a statistically significant
`difference between groups (Po0.05, Student’s t-test).
`
`was different from responsive samples and from each other. In
`AML sample No. 18, CD33 ligation did not induce tyrosine
`phosphorylation or recruitment of Syk or Syk/SHP-1 complex
`formation.
`In AML sample No. 19, CD33 ligation induced
`tyrosine phosphorylation of Syk. However, no Syk/SHP-1 or Syk/
`CD33 complex formation was detected.
`These differences in CD33 signaling confirmed our previous
`findings29 and revealed the importance of not only the presence
`of Syk, but its functional activity. These data altogether prove the
`significance of Syk in CD33 signaling.
`That prompted us to investigate whether or not the response of
`AML cells to GO is also dependent on Syk expression. It is even
`more intriguing as 50% of GO is not conjugated to calichea-
`mycin (package insert), and hence some of its activity is in fact
`due to anti-CD33 mAb signaling. Our results demonstrated a
`strong correlation between the response of AML cells to low
`doses of GO and Syk expression. Higher doses of GO
`diminished differences between Syk-positive and Syk-negative
`samples, possibly owing to the effects of free toxin. Moreover, in
`Syk-positive samples, the number of responders was signifi-
`cantly higher compared to Syk-negative samples. These data
`suggested a correlation between Syk expression and the
`response of AML cells to Mylotarg. To confirm Syk engagement
`in CD33 signaling in anti-proliferative effects of both unconju-
`gated anti-CD33 mAb and Mylotarg, we performed ‘blocking’
`experiments with Syk siRNA. Seventy-two hours after transfec-
`tion, Syk was undetectable by Western blot and HL-60 leukemia
`cells (originally responsive to anti-CD33 mAb cytotoxic activity)
`became unresponsive. The anti-proliferative effect of GO in
`transfected cells also diminished. These data demonstrate the
`importance of Syk in CD33- or GO-mediated cytotoxicity.
`As GO is widely used in the treatment of AML,10 but effective
`only in one-third of patients, understanding the mechanisms of
`its cytotoxicity is important. We hypothesize that determining
`Syk expression before treatment can serve as a biomarker and,
`
`therefore, increase the efficacy of GO or unconjugated anti-
`CD33 mAb.
`In order to augment the anti-proliferative effects of GO and
`anti-CD33 mAb, we considered methods of upregulating Syk
`expression and/or increasing (restoring) its activity. Absence of
`Syk expression, mediated by hypermethylation, is linked to cell
`u