LAB MEETING SUMMARY (8.2.

2011)

Jane presented Drug Screening Unit (DSU) data & work

Current lentiviral short hairpin

RNA resources for stable gene
knockdown (LV shRNA) : In the pipeline :
 Control PIKE
 AKT 1 & 2 & 3 TP53
 ALK EZH2
 AURKA PDGFR α /β
 AURKB KIT
 ERK 1& 2 FGFR1
 IGF1R ERBB2
 MET EGFR
 c-MYC
 n-MYC
 PIK3CA
 PLK1
 RAF1

Chris & Dorine expressed interest in new constructs, will meet separately to discuss

Focus of presentation was on publications coming from DSU work

Multiple pubs in collaboration & two publications in progress strictly from DSU work :

(1)“Down-regulation of PIK3CA induces ERK signalling, stabilisation of c-MYC

and increased IRS-2 expression in rhabdomyosarcoma cell lines providing a
rationale for combination treatments”

(2)“NFκ B signalling constitutively activated in Alveolar rhabdomyosarcoma

resulting in upregulated survivin levels and inhibition of apoptosis”

For publication (1):

Stable knockdown of PIK3CA :
 Causes loss of viability in only ¼ rhabdo cell lines (RMS1, ARMS subtype)
 Does not appear to cause any cell cycle arrest
 Causes increased expression of other PI3K class 1 isoforms
 Increases cellular sensitivity to pan class 1 PI3K inhibitors in ¾ lines (not
RH30)
 Shows increased expression of IRS-2 in ¾ lines (not RH30)
 Increases cellular sensitivity to mTOR inhibition ¾ lines (not RH30)

Conclusion: This data suggests rationale for combining p110α inhibitor w/ mTOR
inhibitor or mTOR inhibitor w/ IGF1R inhibitor
Proof of concept---would be good to see effects of decent IGF1R inhibitor on
PIK3CA KD lines, predict effective in lines that show inc IRS2 expression, is mTOR
+ PI3K inhibition better than mTOR + IGF1R inhibition in this model? In vitro combo
data in parental RMS cell lines?

Stable knockdown of PIK3CA :

 Shows increased phospho-ERK levels
 Are NOT more sensitive to MEK inhibitor despite this
 Pan class 1 PI3K inhibitor & MEK inhibitor show synergistic effects in the
ERMS subtype (RD & RMS-YM cells)
ERMS cells have ~4-fold higher p-ERK relative to ARMS lines w/ PIK3CA KD

ARMS lines lack of sensitivity to MEK inhibitor makes testing these cells for synergy
impractical.

For publication (2):

Stable knockdown of PIK3CA :
 Causes loss of viability in only ¼ rhabdo cell lines (RMS1, ARMS subtype)
 Correlates with stable decrease in surviving expression in RMS1 cells only
 Correlates with inc sensitivity to IKKB inhibitor in ARMS subtype
This data may suggest a requirement for NFkB signalling in ARMS cells

Proposed to do:
o IHC for NFkB on rhabdo TMAs from patient samples
o Data mine the rhadbo expression arrays for inc exp of NFkB pathway genes
o Confirm nuclear localization of NFkB in rhabdo cell lines (confocal, WB)
o Confirm inhibition of NFkB following IKKB inhibitor exposure by confocal
(NFkB relocalisation to cytoplasm)
o In vitro & in vivo combo exposure to pan PI3K and IKKB inhibitors

NEXT:
Preparing to make inducible knockdown lines