Supplementary Figure legends
Supplementary Figure 1. Effect of the combinational therapy of nutlin-3 and valproic
acid on viability of the wt p53 AML cell line MOLM-13. (A) The AML cell line MOLM13 (wild type TP53) was treated with nutlin-3 (0.5 - 10 M) and valproic acid (50 - 1000 M)
alone or in combination for 24, 48 and 72 h (nutlin-3 added for the final 24 h). Cell viability
was detected by DNA specific staining with Hoechst 33342. Viability data were analyzed in
triplicate and data represents the results of three independent experiments (mean ± standard
deviation). (B) Synergism for the interaction of nutlin-3 (5 M) and valproic acid (500 M)
analyzed by Hoechst 33342 was calculated using Bliss independence, in which the fractional
response of a combination of two drugs equals the sum of the two fractional responses minus
their product. From the response to each of the drugs alone, the expected response to the
combination at similar concentrations was calculated. A positive difference between actual
response and expected response was then ascribed to synergy (*** p < 0.001). Error bars
represent standard error of mean. (C) Representative fluorescence microscopy images of
MOLM-13 and peripheral blood lymphocytes stained with DNA specific Hoechst 33342,
following treatment with valproic acid (500 µM), nutlin-3 (5 µM), both or control (DMSO)
for 48 h. White arrows represent apoptotic cells. Images are taken at 40x magnification. (D)
MOLM-13 cells were treated with the combination of nutlin- (2.5 – 5 µM) and valproic acid
(250 – 500 µM) and viability assessed by the colourimetric Alamar blue (24 h) and
bioluminescence based ATP assays (48 h) (*p < 0.05, **0.01 or ***0.001). Synergism was
calculated using Bliss Independence.
Supplementary Figure 2. Effect of the combinational therapy of nutlin-3 and valproic
acid on induction of autophagy. (A) MOLM-13 cells treated with valproic acid (500 M),
nutlin-3 (5 M) or combination of both for 72 h (nutlin-3 for the last 24 h) were incubated
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with Lysotracker Red for 30 minutes and analyzed by flow cytometry. Results are shown as
median fluorescence intensity relative to control (*p < 0.05). (B) MOLM-13 cells were cells
treated with valproic acid (500 M), nutlin-3 (5 M) or combination of both for 48 h (nutlin-3
for the last 24 h), and prepared for analysis by transmission electron microscopy (TEM) for
detection of autophagic features. Arrows indicate autophagosomes.
Supplementary Figure 3. Effect of the combinational therapy of nutlin-3 and valproic
acid on viability of primary AML cells. (A) Example of AML patient sample treated with
nutlin-3 (5 M), valproic acid (500 M) or combination of both for 48 h (nutlin-3 added for
the final 24 h), followed by flow cytometric analysis of viability (Annexin-PI) and data
analysis using FlowJo software version 8.2 (Tree Star, Inc., Ashland, OR, USA). (B)
Differences in means of viability between nutlin-3 (5 M), valproic acid (500 M) or
combination of both for 48 h (nutlin-3 added for the final 24 h) for the pooled patient data
analyzed by Hoechst 33342. Results are given as means ± standard error of mean (* p < 0.05,
n = 34).
Supplementary Figure 4. Effect of the combinational therapy of nutlin-3 and valproic
acid on viability of normal CD34+ cord blood compared to primary AML cells. CD34
positive cells were isolated from 3 different normal cord blood samples (positive selection),
treated with nutlin-3 (5 M), valproic acid (500 M) or combination of both for 48 h (nutlin-3
added for the final 24 h) and flow cytometric analysis of viability was performed (AnnexinPI). The results were compared to the effect in the 10 most sensitive AML patient samples
and the whole cohort of AML patient samples (n = 31). Results are given as means ± standard
error of mean (* p < 0.05, *** p < 0.001).
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Supplementary Figure 5. Development of an optically imageable MOLM-13 AML
xenograft model. (A) Survival curve of NOD-scid (n = 6), NOD-scid 2mnull (n = 6) and
NOD-scid IL2rnull (n = 10) injected i.v with 10 million MOLM-13 or MOLM-13luc cells
following 3, 2.5, or 1.5 Gy irradiation, respectively. While of NOD-scid and NOD-scid
2mnull mice showed variable disease latency with not all animals developing leukemia,
recipient NOD-scid IL2rnull all developed leukemia with median survival of 27 ± 0.8 days.
(B) MOLM-13luc AML development in NOD-scid IL2rnull mice could be monitored
longitudinally with bioluminescence imaging. The figure illustrates results of representative in
vivo bioluminescence imaging of dorsal and ventral aspects of an animal injected with 10
million MOLM-13luc cells. Images were acquired 10 minutes following injection of 150
mg/kg of D-luciferin i.p, and performed once per week for 3 weeks and on day 24 after cell
injection. Color bars represent photon counts per raster scan point (1 mm2) per second. (C)
Quantification of total photon counts per second, per whole-body, region of interest plotted
versus time (n = 4 mice). Inset shows total photon counts per second for dorsal and ventral
regions of interest. (D) Following necropsy, liver, spleen, lymph nodes, femur, lungs and GItract were resected out and imaged. All organs showed bioluminescent signals, which was
particularly intense at the cecum-ileum junction (white arrow) and also in the appendix. (E)
(Middle panel) Photo comparing appendix and upper part of the large intestine of healthy
NOD-scid IL2rnull mouse and MOLM-13luc xenografted NOD-scid IL2rnull in moribund
condition. Hematoxylin and eosin staining of representative histology sections of the colon
and ileum confirmed MOLM-13 cell infiltration. Microscopy images are 10x and (inset) 40x
magnification.
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