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MELK 3-11-2016
Supplementary Figure 1- MELK expression is absent in normal tissues but present in breast
tumors. Serial analysis of gene expression (SAGE) analysis of MELK RNA expression in normal
and tumor tissue demonstrates MELK expression in cancerous tissue, including breast, with
limited expression in normal tissue. Normalized tag counts depicted on the x-axis with tissue
type depicted on the y-axis (A).
Supplementary Figure 2- MELK RNA is more highly expressed in ER-negative and basal-like
human breast cancer cell lines compared to ER-positive and luminal-like breast cancer cell lines.
MELK expression is significantly elevated in basal A and basal B breast cancer cell lines as
measured by Affymetrix gene expression profiling from a publically available dataset (Neve et
al.) (A). Breast cancer cell line is depicted on the x-axis and mean centered log2 expression on
the y-axis. Hormone receptor and intrinsic subtype status is also depicted. ER-negative breast
cancer cell lines have higher expression of MELK, as do the breast cancer cell line subtypes
basal A and basal B which are enriched for triple-negative breast cancer cell lines (B). This was
confirmed by qRT-PCR looking at MELK RNA expression in 14 breast cancer cell lines (C).
The expression of MELK in 14 breast cancer cell lines (9 ER-negative and 5 ER-positive) was
measured using Q-RT-PCR analysis. Data is depicted as relative fold change normalized to
cyclophilin. Data are represented as mean ± SD; Supplementary Figure 3- MELK protein is
more highly expressed in ER-negative and basal-like human breast cancer cell lines compared to
ER-positive and luminal-like breast cancer cell lines.
MELK protein expression is significantly
elevated in ER-negative breast cancer cell lines compared to ER-positive breast cancer and
normal mammary epithelial cell lines. MELK protein in 15 cell lines (1 normal, 6 ER-positive,
and 9 ER-negative) was measured by western blotting. A representative blot is shown (A).
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MELK protein expression is significantly elevated in ER-negative as compared to ER-positive
breast cancer cell lines (B-C). There is a strong correlation between MELK RNA expression
(measured by qRT-PCR) and protein expression (measured by western blotting) levels (D).
Western blotting was performed at least three times with three difference batches of breast
cancer cell lines grown under basal growth conditions. Data is depicted as relative fold change
normalized to MCF-7 cell line expression. Data are represented as mean ± SD; Supplementary
Figure 4- SF2Gy is decreased after MELK knockdown without significant toxicity in TNBC
lines. Surviving fraction after 2 Gy of radiation is decreased after MELK knockdown in MDAMB-231 cells (A) and BT549 cells (B) with limited toxicity. Conversely, radioresistance is
conferred on MCF-7 cells by the overexpression of MELK with increase in SF-2Gy values (C).
Clonogenic survival using additional siRNA constructs from a different vendor (siMELK#3 and
siMELK#4) again demonstrates radiosensitization MDA-MB-231 (D) and BT-549 (E) triplenegative breast cancer cell lines using two independent siMELK constructs with enhancement
ratios (ER) of 1.26-1.50 suggesting specific, and not off-target, effects. Data are represented as
mean ± SD; Supplementary Figure 5- MELK inhibition using genetic and pharmacologic
techniques demonstrates significant radiosensitization with MELK knockdown or kinase
function inhibition. Clonogenic survival demonstrates inducible shMELK expression
significantly radiosensitizes MDA-MB-231 triple-negative breast cancer cell lines using two
independent shMELK constructs with enhancement ratios (ER) of 1.44-1.52 with a significant
decrease in surviving fraction after 2 Gy (A). Similarly, inhibition of MELK kinase function
using the MELK inhibitor OTSSP167 at various concentrations causes significant
radiosensitization with ER or 1.61-1.68 (B). Similar results were seen with MELK inhibition in
BT549 cells (data not shown). Data are represented as mean ± SD.
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Supplementary Figure 6- MELK knockdown or inhibition does not significantly impact
proliferation in several cell lines. Proliferation assays demonstrated that MELK knockdown
delays growth in MDA-MB-231 cells (B) but not BT-549 (A) or MCF-7 cell lines (E). Similarly,
MELK inhibition with various concentrations of the MELK inhibitor OTSSP167 has a similar
effect on growth in these three cell lines (B,D,F); Supplementary Figure 7- MELK knockdown or
overexpression does not significantly impact rates of apoptosis in several cell lines by western
blotting. Cleaved PARP was used to assess the effect of MELK knockdown or overexpression on
the amount of apoptosis. There was no significant changes in cleaved PARP levels with either
MELK knockdown or overexpression in 3 different cell lines; Supplementary Figure 8- MELK
knockdown or inhibition does not significantly impact rates of apoptosis in several cell lines by
flow cytometry. Dual PI and Annexin V staining was used to assess the amount of early and late
apoptosis after MELK knockdown or inhibition. In most cell lines there was no significant
change in wither early (A) or late (B) apoptosis with either MELK knockdown or inhibition.
Representive data from one time point is represented in (C). Error bars represent +/- SD of at
least three independent experiments.
Supplementary Figure 9: MELK is implicated in the DNA damage response pathway. Gene set
enrichment analysis (GSEA) identifies the top positively (A) and negatively (B) associated
concepts. For all depicted positively and negatively associated GSEA concepts the FDR and Pvalues were <0.001. The top 10 N-enrichment scores for both groups are depicted, with orange
highlighting concepts highlighted in C and D. GSEA enrichment scores for 2 of the top 5
positively correlated concepts are depicted (C, D); Supplementary Figure 10: MELK is
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implicated in the DNA damage response pathway. Genes with the highest correlation to MELK
expression in the TCGA data set are enriched for cellular functions that include DNA repair, cell
cycle regulation, and cell survival as well as canonical pathways that include BRCA1 in DNA
damage response and G2/M DNA damage checkpoint repair- analysis done using Ingenuity
pathway analysis (A). The top canonical pathway, ‘role of BRCA1 in DNA damage response’ is
depicted (B) with the genes whose expression is significantly positively correlated with MELK
expression in TCGA depicted in red.
Supplementary Table 1- Enhanced combination effect at different time points during treatment
with combination radiation and MELK knockdown or inhibition. Fractional product method was
used to compare the effects of combination treatment. Fractional tumor volumes were calculated
for the various treatment arms and various time points and combined.
The indices for
combination therapy with radiation and MELK knockdown/inhibition were >1, indicating a
synergistic interaction between the drugs; Supplementary Table 2- Clinical characteristics of the
patients and tumors in the Wang dataset; Supplementary Table 3- Clinical characteristics of the
patients and tumors in the Servant dataset
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