1. Discovering new therapeutic targets in GIST
Welcome to the GIST conference here at OHSU and I am so excited to speak to you again this year. Today I will tell you about 2 different projects we are working on in the lab. One in Kit-mutant GIST the other in PDGFRA mutant GIST.
Lillian Klug, PhD
Post-doctoral Scholar, Heinrich Lab
GCRF conference | May 8, 2018

2. Gastrointestinal stromal tumor (GIST) is a unique disease
GIST is caused by mutations in DNA
Mutations in DNA can turn on the activity of proteins that are normally not active in adults= cancer growth
Cancer growth can be stopped if mutant proteins can be turned off

3. Mutations seen in GIST
The majority of mutations in GIST are KIT mutations, but mutations in PDGFRA and SDH subunit and a few other genes can drive GIST as well.
Today I will talk about KIT-mutant GIST and PDGFRA mutant, specifically D842V mutant GIST.

4. Opening the door to a GIST cure
So our ultimate goal is a cure for GIST, and while we have successful treatments now, there is still room for improvement.
I want to walk you through how we approach GIST treatment and some of the biology that can present problems and how it prevents our current treatments from being curative.
To do this, I want to use this metaphor of a door to represent the protein KIT that is often mutated in GIST.

5. KIT
In normal adult cells, KIT plays a fairly passive role. That is represented by the door being open.

6. mutation
KIT
In normal adult cells, KIT plays a fairly passive role. That is represented by the door being open.
Random mutations can strike which cause abnormal activity.

7. KIT KIT exon 11 deletion mutation
In this metaphor, abnormal activity is represented by a closed locked door, which leads to cancer growth.
Fortunately, from the research we have done, we can profile these different mutations to determine treatment with specific drugs like Gleevec.

8. KIT
… and open the door. Tumors will shrink and patients can survive for a long time on Gleevec.

9. KIT
However, often at some point during Gleevec therapy, another mutation can spontaneously occur creating another lock.
But more universal drugs, like sutent, have been developed to unlock these mutations…
and while we have a third KIT inhibitor option, stivarga, this problem continues…

10. KIT
… at some point the locks become too complex for any one key to be able to open all of them. This represents drug-resistance.
DRUG-RESISTANCE

11. ?
What is next?
KIT
So patients can go from Gleevec to Sutent to Stivarga as their disease progresses, but once resistance occurs after 3rd line therapy, we have few drug treatment options left for patients.
So this is where my project picks up. It asks, what is next? What can we do in the face of drug resistance?

12. KIT
We know that this open door is still the goal, so what other ways can we achieve this?

13. GIST cell models
To investigate this, we use GIST cells that have come from human GIST that we can grow in plastic flasks.
*GIST cell lines that grow in the lab have KIT mutations

14. GIST cell models
These GIST cells behave like human tumors and we can apply drugs like Gleevec and see cells that have sensitive KIT mutations die and ones with resistant mutations remain viable. This is what we would expect to happen in patients.

15. Targets in KIT-mutant cells
GIST430
ALK
EPHB6
ROS1
SYK
DDR1
DDR2
PTK7
TIE
KIT
LMTK3
PTK2
EPHA6
TYK2
After doing tons of experiments in 3 different KIT-mutant cell lines, I whittled down the list of candidates to a few in each cell lines and looked at those that were shared to identify ones that are important regardless of mutation.
The only targets shared by all 3 were KIT, which we already know is a good target, because it is the target of Gleevec and one other protein called LMTK3
EPHA4
FGFR3
MSTR1
ZAP70
TYK2
PTK2
EPHA6
NRAS
FLT1
KIT
LMTK3
KIT
LMTK3
ABL2
TP53RK
FGFR1
LTK
LTK
GIST-T1
MaMel

16. KNOCKDOWN OF LMTK3 KILLS CELLS WITH KIT RESISTANCE MUTATIONS
Drug-resistant
LMTK3
As I began to study LMTK3 in GIST cells, I found that inhibiting LMTK3 can kill all the KIT mutant cells, regardless of mutation. It was able to reduce the growth of both Gleevec sensitive and Gleevec resistant cells, suggesting that this protein could be a promising therapeutic target. And we are continuing to study LMTK3 to understand exactly what it does in GIST cells.
From what I have found, is that LMTK3 seems to play an important role in these GIST cells and by reducing LMTK3 levels in these cells, we don’t necessarily unlock the locks, but instead…

17. Mutations seen in GIST
The second project I am working on concerns PDGFRA mutant GIST.

18. PDGFRA
D842V
PDGFRA is another protein that can take on abnormal activity when mutated, like KIT.
There is one mutation in particular that is most common and the most hard to treat, called D842V. None of the clinically available inhibitors can unlock this mutation.

19. Creating a PDGFRA D842V GIST model
No cell lines
GIST430
GIST-T1
GIST882
GIST48
GIST54
GIST62
We would love to study this kind of GIST in the lab, but, while there are good models of KIT-mutant GIST. No models of PDGFRA D842V GIST exist.
So we decided to try to create one.

20. Creating a PDGFRA D842V GIST model
BLU-285
“Avapritinib”
It is important to have a cell model of D842V GIST, because there is a new drug, called BLU-285, or avapritinib, that is not only able to inhibit KIT-mutant GIST, but…
STOP

21. Creating a PDGFRA D842V GIST model
STOP
BLU-285
“Avapritinib”
It can also inhibit PDGFRA-mutant GIST, specifically D842V
This drug has entered clinical trials with really promising results.
However, we don’t know about resistance to this drug yet, since it is so new.
We would like to thoroughly investigate the way BLU-285 works in these cells, but to do that we need a model we can use in the lab.

22. Creating a PDGFRA D842V GIST model
- KIT ex 11
GIST430
GIST-T1
GIST882
GIST48
GIST54
GIST62
To create a PDGFRA D842V model, we took a KIT mutant cell line and added D842V to it and took out KIT. Because PDGFRA is so similar, to KIT, it was able to take over driving the growth of these cells.

23. Creating a PDGFRA D842V GIST model
GIST-T1 + D842V
We can then use these cells with D842V in assays and compare the effects of imatinib to those with BLU-285.
Like we expect, the D842V cells are resistant to imatinib, but sensitive to BLU-285. This mirrors what we see in patients in the clinic.
IMATINIB
BLU-285

24. Creating a PDGFRA D842V GIST model
GIST-T1
GIST882
GIST48
GIST54
GIST62
GIST-T1+D842V
Now that we have a D842V model, we can begin investigating resistance and testing even more drugs to overcome resistance in the hopes of getting ahead of resistance to help patients!

25. THANK YOU!
Questions??
With that I would like to thank you all for being here. It is really a pleasure to share my research with you all.
And I am happy to take any questions you might have!