1. • In cancer, specific muta0ons and DNA altera0ons are closely associated with oncogenic
transforma0on and tumor progression.
• Recent studies highlight the importance of epigene0c changes to cancer, in which the
DNA sequence remains unchanged but the biology of the tumor is affected by structural
altera0ons to the DNA and its associated machinery. In human cells, DNA is coiled
around octomeric proteins called histones forming what is called chroma0n. The
structure of chroma0n can be remodeled by adding or removing small molecules like
methyl groups onto the histone proteins (Figure 1). This results inhibi0ng or ac0va0ng
transcrip0on of select genes.
• Drug-specific therapies have been proposed that might counter-act these structural
altera0ons so as to limit the rampant growth of cancer cells.
• Glioblastomas (GBM) are highly aggressive brain tumors that exhibit different molecular
subtypes with variable prognoses:
• Proneural (PRO) tumors generally have bePer prognosis
• Mesenchymal (MES) tumors are generally more resistant to standard
therapies
• GBMs have been shown to transi0on from the PRO phenotype to the MES phenotype. A
similar transi0on occurs in other solid tumors and has been called the “epithelial to
mesenchymal transi0on” (EMT).
• This transi0on in GBM has been linked to specific transcrip0on and signaling pathways
including STAT3 and NF-kB. The MES phenotype is also characterized by high levels of
CD44.
• In other solid tumors including prostate cancer, certain histone modifica0ons are
strongly correlated with tumor grade, EMT, and disease recurrence.
• The rela0onship between the epigene0c modifica0on, transcrip0on factors and tumor
molecular subtypes in GBM remains unknown.
• Lysine (K) demethylase 1A (LSD1) is a protein known to act specifically on lysines 4 and 9
of histone 3. LSD1 also has a known role in cell differen0a0on. It plays cri0cal role for
epigene0c modifica0on and gene expression.
Combined targeted inhibi/on of STAT3 and the
epigene/c regulator LSD1 alters prolifera/on and
molecular phenotype in glioblastoma tumor cells
John Peterson, University of Utah | Howard Colman, MD, PhD, University of Utah
Study conducted with funding from a U of U Undergraduate Research and
Crea0ve Opportunity Grant and lab assistance from the U of U
Department of Biology.
John Peterson
University of Utah
Biology Department
john.peterson@hci.utah.edu
I. Introduc0on
• To inves0gate the role of transcrip0onal and epigene0c alterna0ons, we u0lized the GSC20
(MES) and GSC23 (PRO) GBM stem cell lines derived from individual human tumors. Serum
exposure at 10% concentra0on was used to induce the transi0on to the MES phenotype in
GSC23 cells as evident by the drama0c increase in PY-STAT3 and CD44 (Figure 2A). The cells were
treated with S31-201 (a STAT3 inhibitor), and HCI2509 (an LSD1 inhibitor) individually and in
combina0on to target transcrip0on factors or epigene0c regulators of cell differen0a0on. Levels
of methylated histone proteins were measured by Western Blot (Figures 4A and 4B).
• Cell prolifera0on assays were also performed in GSC20 to compare the cell growth of MES cells
in the presence of 2509 and/or S31-201 (Figure 3A). The level of cell prolifera0on was measured
by comparing the levels of fluorescence in prolifera0on assay and the colony numbers grew in
soh agar assay (Figure 3B).
II. Methods
III. Results
Conclusions: Our results demonstrate that combined inhibi0on of the STAT3 signaling pathway and
and LSD1 have addi0ve or synergis0c effects on expression of PRO and MES markers, colony
forma0on and prolifera0on, and epigene0c modifica0ons in GBM. At the epigene0c level, our data
suggest that decrease in the level H3K4me3 is associated with MES transi0on. These findings
indicate poten0al therapeu0c strategies to inhibit MES transi0on and sensi0ze GBM cells to current
therapies by targe0ng both the transcrip0on machinery, specifically STAT3, and the methyla0on of
lysine 4 of histone 3 via LSD1.
Future Direc/ons: We are currently inves0ga0ng the expression of these biomarkers in pa0ent
tumor samples by immunohistochemistry (Figures 5 and 6). We aim to validate our in vitro
observa0ons in the human tumors and determine the associa0on of specific biomarkers with
respec0ve pa0ent’s genomic and survival data. In this way we hope to bePer connect a pa0ent to
the most effec0ve cancer therapy for their tumor based on the levels of these key biomarkers.
IV. Conclusions & Future Direc0ons
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Figure 1
Figure 2A Figure 2B
DMSO 5uM 10uM 15uM
HCI2509Figure 3A
0 2 4 6 8 1 0
0
1
2
3
d a y s
570/595ratio
D M S O
2 5 0 9 (5 u M )
2 5 0 9 (1 0 u M )
2 5 0 9 (1 5 u M )
2 5 0 9 (2 0 u M )
2 5 0 9 (2 5 u M )
Figure 3B
• Western Blot showing levels of MES biomarkers in GSC23 and GSC20. The addi0on of 150 μM
S31-201 to GSC23 exposed to serum led to a no0ceable decrease in levels of PY-STAT3 sugges0ng
that the transi0on to the MES phenotype had been inhibited.
• When 10 μM of HCI2509 is added, a similar decrease in PY-STAT3 levels is seen. When the two
drugs are used in combina0on at their respec0ve concentra0ons, STAT3 is completely blocked
and CD44 expression reduced in the presence of serum (10%) as shown above (Figure 2A).
• When the GSC20 cells were exposed to HCI2509 alone, levels of H3K4me3, H3K9me2 and Olig2
increase, while levels of CD44 and PY-STAT3 decrease (Figure 2B)—both of which are considered
biomarkers for the MES phenotype.
• Cell prolifera/on of GSC20 cells in the presence of HCI2509 with quan/fica/on.
GSC20 prolifera0on was inhibited in the presence of 15 μM HCI2509 as shown by a decrease
in soh agar colony numbers (Figure 3A) and quan0fica0on of cell number by fluorescence
(Figure 3B). At the higher concentra0ons of 20 μM and 25μM, cell prolifera0on essen0ally
stops and this matches the soh agar assay.
GSC20
+
15
100
15
100
CD44
LSD1 dark
LSD1
EZH2
PY-STAT3
Olig2
H3K4me3
H3K9me2
GAPDH
DMSO
2509 (uM)
S31-201 (uM)
Figure 4A Figure 4B
• Western Blots showing levels of epigene/c biomarkers in GSC20 cells. Trea0ng GSC20 with
S31-201 increases their levels of H3K9me2. A combina0on treatment of HCI2509 and S31-201
leads to a addi0ve or synergis0c increase in H3K4me3, and H3K9me2 (Figure 4A). This same
combina0on treatment also increases levels of the PRO biomarker Olig2 while simultaneously
decreasing CD44 and PY-STAT3 (Figure 4A).
Figure 5:
GBM sec0on from
pa0ent stained for CD44
(as shown by brown dye).
Figure 6:
GBM sec0on from pa0ent
stained for Olig2 (as
shown by brown dye in
nuclei).