Dr Kathy Gately is a Clinical Scientist at St James's Hospital and Clinical Senior Lecturer at Trinity College Dublin. Dr Gately's research interests are Drug Resistance Mechanisms in Non-Small Cell Lung Cancer, Liquid Biopsy and Organoid models.

1. Targeting PIM Kinase to Overcome
Drug Resistance in NSCLC
Kathy Gately PhD
Clinical Scientist & Clinical Senior Lecturer
Thoracic Oncology Research Group
St. James’s Hospital

2. PIM Kinases
• PIM proteins are a family of serine/threonine
kinases.
• PIM1, PIM2 & PIM3 are located on
chromosome 17, the X chromosome and
chromosome 15 respectively.
• Play a role in cell cycle progression,
metabolism, inflammation and immune
evasion.
• PIM kinases are constitutively active & their
activity is regulated at the level of transcription,
translation, and proteasomal degradation.
• The JAK/STAT pathway is one of the key
upstream transcriptional regulators of PIM
kinases.

3. PIM Kinase – Involved in Hallmarks of Cancer

4. Elevated PIM Kinase Correlates to Poor Survival in
Hematological & Solid Tumours
PIM1
PIM2
PIM3
αβ- tubulin
34 kDa
40 kDa
34 kDa
35 kDa
55 kDa
A. B. No PIM1 Weak PIM1 Strong PIM1 C.
.
Overall PIM1 positive patients had a median survival time of 23.67 months versus 37.32 months in PIM1 negative group (n=134, Log-rank test p=0.056).

5. • Co-regulator of c-Myc
• Regulator of c-MET
• Regulator of mTOR
• Activated in response to
Chemotherapy, Radiotherapy &
Targeted Therapies in NSCLC
PIM Kinase -Regulates a Network of Signaling
Pathways Critical for Tumorigenesis & Development

6. PIM Kinase Causes Resistance to PI3K Inhibition
Pharmacol Ther. 2020 Mar;207:107454.

7. Activated PIM Kinase a Resistance Mechanism
to PI3K-mTOR Inhibition in NSCLC

8. Development of a Panel of GDC-0980 Resistant NSCLC Cell Lines
Sci Rep. 2018 Jan 26;8(1):1652

9. Development of Resistant Cell Line Models In-depth Characterisation of Resistant Models Activated PIM kinase expression in all 3 Models
Investigating pipeline drugs/ Biomarker Discovery Validation of miRNA signature Validation of Dysregulated Protein Expression
Microarray
mRNA/miRNA
Gene
Name
H1975GR
vs H1975P
(Month 4)
H460GR vs
H460P
(Month 5)
A549GR vs
A549P
(Month12)
PIM1 19.8 14.3 78.6
PIM2 3.3 2.08 1.29
PIM3 31.98 10.9 17.89
MYC 25.65 13.9 30.2
MET 6.06 5.4 2.6
Characterisation of GDC-0980 resistant H1975GR cell line
Cancers (Basel). 2021 Apr 29;13(9):2139

10. Validation of a PIM1/MYC associated miRNA signature
in PI3K/mTOR inhibitor resistant H1975GR cells
miR-17-5p
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
2.0
2.5
**
Relative
gene
expression
(normalised
to
mean
reference
genes)
miR-20a-5p
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
2.0
*
Relative
gene
expression
(normalised
to
mean
reference
genes)
miR-18a-5p
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
Relative
gene
expression
(normalised
to
mean
reference
genes)
miR-19a-3p
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
2.0
Relative
gene
expression
(normalised
to
mean
reference
genes)
miR-19b-3p
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
2.0
2.5
*
Relative
gene
expression
(normalised
to
mean
reference
genes)
miR-18b-5p
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
Relative
gene
expression
(normalised
to
mean
reference
genes)
miR-206
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
2.0
Relative
gene
expression
(normalised
to
mean
reference
genes)
*
miR-486-5p
H
1
9
7
5
P
H
1
9
7
5
G
R
0
1
2
3
Relative
gene
expression
(normalised
to
mean
reference
genes)
miR-203a
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
Relative
gene
expression
(normalised
to
mean
reference
genes)
*
miR-328-3p
H
1
9
7
5
P
H
1
9
7
5
G
R
0
1
2
3
4
Relative
gene
expression
(normalised
to
mean
reference
genes)
(A) (B)
miR-15b-5p
H
1
9
7
5
P
H
1
9
7
5
G
R
0.0
0.5
1.0
1.5
2.0
Relative
gene
expression
(normalised
to
mean
reference
genes)
*
miR-17 miR-18a miR-19a miR-20a miR-19b-1 miR-92a-1
miR-106a miR-18b miR-20b miR-19b-2 miR-92a-2 miR-363
miR-106b miR-93 miR-25
miR-106a/363
miR-106b/25
miR-17/92 family
paralogues
Cancers (Basel). 2021 Apr 29;13(9):2139

11. (B)
(A)
IC50 (nM)
PI3Kα mTOR PIM1 PIM2 PIM3
IBL-301 2.5 134 18.2 13.6 3.17
**
*
Efficacy of IBL-301 in H1975GR versus H1975GP cell lines
Cancers (Basel). 2021 Apr 29;13(9):2139

12. (A)
(C)
αβ-tubulin
UT 250 500 UT 250 500
CaLu-6
6 hr 24 hr
αβ-tubulin
αβ-tubulin
c-Myc
c-Myc
c-Myc
H1975
H1838
55 kDa
60 kDa
55 kDa
60 kDa
55 kDa
60 kDa
6 hr 24 hr
2 hr
H1975
PIM1
Phospho-4EBP1
(Thr36/47)
Phospho-eiF4B
(Ser422)
αβ-tubulin 55 kDa
80 kDa
80 kDa
40 kDa
(D)
(B)
p
E
R
K
p
S
T
A
T
1
p
S
T
A
T
3
p
A
k
t
T
h
r
3
0
8
p
A
k
t
S
e
r
4
7
3
p
A
M
P
K
a
p
S
6
p
m
T
O
R
p
B
A
D
p
7
0
S
6
K
P
R
A
S
4
0
p
5
3
p
3
8
S
A
P
K
/
J
N
K
P
A
R
P
C
a
s
p
a
s
e
3
G
S
K
-
3
b
0.0
0.5
1.0
Untreated BEZ235 IBL-301
Fold
Change
in
protein
levels
(relative
to
untreated
control)
IBL-301 Inhibits Intracellular Signalling in NSCLC Cell Lines
Cancers (Basel). 2021 Apr 29;13(9):2139

13. Effect of BEZ235 vs IBL-301 on Intracellular Signaling
in Patient Derived Explant (PDE) NSCLC Models
(C)
Untreated
BEZ235
IBL-301
(A) (B)
Untreated BEZ235 IBL-301
-5,000
0
5,000
10,000
15,000
20,000
MCP-1
NSCLC ex-vivo
pg/mL
**
(B)
Cancers (Basel). 2021 Apr 29;13(9):2139

14. Summary
• PIM kinases & cMYC are activated in response to PI3K-mTOR inhibition in EGFR
TKI resistant, PIK3CA mutated NSCLC cells
• Co-targeting PI3K and PIM kinase may overcome resistance to PI3K inhibition
providing a more durable response to treatment
• Identification of a PIM1 & MYC associated miRNA signature that predicts
PI3K/mTOR inhibitor resistance
• Pro-inflammatory chemokine MCP-1 is elevated in PI3K/mTOR inhibitor
resistance & inhibited by IBL-301

15. Activated PIM Kinase a Resistance Mechanism
to EGFR TKIs in NSCLC

16. Pathway Crosstalk Driving Resistance to EGFR TKIs
PIM kinase - a common node within these signalling pathways and its activation has
emerged as a significant mechanism of resistance to several pathway inhibitors.

17. In silico analysis - Microarray study to understand acquired resistance to
EGFR-targeted therapy in lung cancer (E-GEOD-38310 )

18. -1 2 -1 0 -8 -6 -4 -2
-5 0
0
5 0
1 0 0
1 5 0
N o rm a liz e o f IB L -3 0 1
L o g 1 0 [c o m p o u n d ]M
V
ia
b
ili
t
y
(
%
;
n
o
r
m
a
li
s
e
d
R
F
U
)
H C C 8 2 7
H C C 8 2 7 G R `
H C C 8 2 7 G R C lo n e 3
PIM1 Expression & Efficacy of IBL-301 in HCC827/HCC827GR & Clone3 Cell Lines

19. Development of Erlotinib-Resistant HCC827 Cell line Models
(a) Erlotinib resistance was developed over 4 months via the addition of increasing
concentrations of erlotinib (10nM - 5µM) until a final concentration of 5μM
(a) To further characterise the different resistant clones present in the HCC827ER cell
population HCC827ER cells were diluted to approximately 5 cells/ml and grown in
erlotinib. 14 subclones were isolated and characterised, 6 clones were MET amplified
(including Clone 3) and 8 clones were more EMT-like (including Clone 10).
b
Oncogenesis. 2017 Apr 3;6(4):e307

20. PIM1, PIM3, MET & c-Myc Expression
in HCC827P vs HCC827ER Cell Lines
MET
c-Myc
PIM-1
PIM-3
/ tubulin
140kDa
60kDa
44kDa
34kDa
35kDa
55kDa
HCC827P
HCC827ER
HCC827P
HCC827ER
HCC827P
HCC827ER
Activated MET, PIM1, PIM3 & c-Myc expression in erlotinib resistant HCC827ER cells
compared to erlotinib sensitive HCC827P cells.

21. Immunofluorescence staining of MET, PIM-1 & PIM-1/MET
expression in HCC827ER cells
(a) MET expressed in some clones within the mixed population of erlotinib resistant cells (HCC827ER)
(b) PIM1 is expressed in all cell populations in the HCC827ER cell line
(c) Dual staining of MET and PIM1 in the HCC827ER cell line
A B C

23. Efficacy of PI3K/mTOR/PIM inhibitor IBL302 in HCC827P/ER, Clone
3 & Clone 10 cell lines
(a) Drug dose response curves examined the efficacy of AUM302 in HCC827P versus
HCC827ER cells using the Cell Titre Blue assay (Promega). Cell viability was measured
after drug treatment for 72hr (IC50=255.3nM vs. 179.8nM, n=3)
(a) Drug dose response curves, examined the efficacy of AUM302 in HCC827ER Clone 3
(MET amplified) versus HCC827ER Clone 10 (EMT amplified). Cell viability was
measured after drug treatment for 72hr (IC50=106nM vs. 23.53nM, n=3).

24. Effect of pan-PIM inhibitor AZD1208 alone/+ Erlotinib on
Protein Kinase Phosphorylation in Clone 3 (MET)

25. Summary
• PIM1/2/3 & cMyc are activated in EGFR TKI Resistant HCC827ER cells
• PIM1 & c-MYC are activated in EMT like Clone 10
• pSTAT3 is activated in HCC827ER cells & Clone 3 & Clone 10
• HCC827ER, Clone 10 (EMT) and Clone 3 (MET) are more sensitive to IBL-302
than HCC827P cells
• Both in-vitro and ex-vivo studies are ongoing to determine the efficacy of PIM
targeted therapy in EGFR mutant NSCLC

26. Thank You
• Dr Gillian Moore
• Dr Clara Lightner
• Dr Susan Heavey
• Dr Anne Tranberg Madsen
• Dr Michelle Simone Clement
• Ms Nathalie Simon
Collaborators:
• Prof Boe Sørensen - Aarhus University Hospital, Denmark
• Prof Andrew Kraft - University of Arizona Cancer Center
Funding:
• Enterprise Ireland Innovation Partnership