PTEN mutant NSCLC require ATM to suppress pro-apoptotic signalling and evade radiotherapy

TL;DR: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage, which creates an exploitable bottleneck and low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.

Abstract: Background: Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy. Methods: Using CRISPR genome editing, we deleted PTEN in a human tracheal stem cell-like cell line as well generated primary murine NSCLC, proficient or deficient for Pten, in vivo. These models were used to verify the impact of PTEN loss in vitro and in vivo by immunohistochemical staining, western blot and RNA-Sequencing. Radiation sensitivity was assessed by colony formation and growth assays. To elucidate putative treatment options, identified via the molecular characterisation, PTEN pro- and deficient cells were treated with PI3K/mTOR/DNA-PK-inhibitor PI-103 or the ATM-inhibitors KU-60019 und AZD 1390. Changes in radiation sensitivity were assessed by colony-formation assay, FACS, western-blot, phospho-proteomic mass spectrometry and ex vivo lung slice cultures. Results: We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model. Conclusion: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.

Summary

Introduction

• For cell 97 detachment Trypsin (Sigma Aldrich) was used.
• Result files were filtered 395 for the included genes to create pathway specific visualizations.
• The authors study suggests that tumors harbouring a loss of function mutation in PTEN can 930 be therapeutically addressed by irradiation in combination with ATM inhibition.
• BioRxiv preprint 26 h after radiation with 8 Gy (dashed lines) or without radiation (continuous 1108 lines) (dead cells stained with trypan blue excluded from analysis).

Full text

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PTEN mutant NSCLC require ATM to suppress pro-apoptotic signalling and evade
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radiotherapy
2
3
Thomas Fischer
1,2,3
, Oliver Hartmann
2,3,*
, Michaela Reissland
2,3*
, Cristian Prieto-
4
Garcia
2,3
,
Kevin Klann
4
, Christina Schülein-Völk
5
, Bülent Polat
1,6
, Elena Gerhard-
5
Hartmann
6,7
, Mathias Rosenfeldt
6,7
,
Christian Münch
4
,
Michael Flentje
1
& Markus E.
6
Diefenbacher
2,3,
ŧ
7
8
9
1
Department of Radiation Oncology, University Hospital Würzburg, Würzburg, Germany
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2
Protein Stability and Cancer Group, University of Würzburg, Department of Biochemistry and Molecular Biology,
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Würzburg, Germany
12
3
Mildred Scheel Early Career Center, Würzburg, Germany
13
4
Protein Quality Control Group, Institute of Biochemistry II, Goethe University Frankfurt, Germany
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5
Core Unit High-Content Microscopy, Biocenter, University of Würzburg, Germany
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6
Comprehensive Cancer Centre Mainfranken, Würzburg, Germany
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7
Institute for Pathology, University of Würzburg, Germany
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*Equal contribution
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ŧ
Corresponding author
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Running Title: PTEN loss establishes radiotherapy resistance
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Keywords: PTEN, ATM, IR, NSCLC, Radiotherapy, Cancer. DNA-PK, PI3K.
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*Corresponding Author: Dr. Markus E. Diefenbacher. Lehrstuhl für Biochemie und
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Molekularbiologie, Biozentrum, Am Hubland, Würzburg, 97074, Germany. Phone:
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0931 31-88167; Fax: 0931 31-84113; E-mail: markus.diefenbacher@uni-
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wuerzburg.de
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Conflict of Interest: The authors declare no potential conflicts of interest.
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.CC-BY 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 25, 2021. ; https://doi.org/10.1101/2021.07.24.453632doi: bioRxiv preprint

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Abstract
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Background
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Despite advances in treatment of patients with non-small cell lung cancer, carriers of
50
certain genetic alterations are prone to failure. One such factor frequently mutated, is
51
the tumor suppressor PTEN. These tumors are supposed to be more resistant to
52
radiation, chemo- and immunotherapy.
53
54
Methods
55
Using CRISPR genome editing, we deleted PTEN in a human tracheal stem cell-like
56
cell line as well generated primary murine NSCLC, proficient or deficient for Pten, in
57
vivo. These models were used to verify the impact of PTEN loss in vitro and in vivo
58
by immunohistochemical staining, western blot and RNA-Sequencing. Radiation
59
sensitivity was assessed by colony formation and growth assays. To elucidate
60
putative treatment options, identified via the molecular characterisation, PTEN pro-
61
and deficient cells were treated with PI3K/mTOR/DNA-PK-inhibitor PI-103 or the
62
ATM-inhibitors KU-60019 und AZD 1390. Changes in radiation sensitivity were
63
assessed by colony-formation assay, FACS, western-blot, phospho-proteomic mass
64
spectrometry and ex vivo lung slice cultures.
65
66
Results
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We demonstrate that loss of PTEN led to altered expression of transcriptional
68
programs which directly regulate therapy resistance, resulting in establishment of
69
radiation resistance. While PTEN-deficient tumor cells were not dependent on
70
DNA-PK for IR resistance nor activated ATR during IR, they showed a significant
71
dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via
72
KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in
73
PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic
74
ex vivo tumor model.
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Conclusion
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PTEN tumors are addicted to ATM to detect and repair radiation induced DNA
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damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we
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show that low concentration of ATM inhibitor is able to synergise with IR to treat
80
PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.
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.CC-BY 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 25, 2021. ; https://doi.org/10.1101/2021.07.24.453632doi: bioRxiv preprint

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MATERIAL AND METHODS
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Cell lines
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Human BEAS-2B and HEK 293T cell lines was obtained from ATCC. Cells were
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maintained in high-glucose DMEM (Sigma Aldrich) supplemented with 10% FBS
95
(Capricorn Scientific) and 1% Pen-Strep (Sigma Aldrich) 1% Glutamin (Sigma
96
Aldrich) at 37°C in 5% CO
2
on 10 cm dishes (Greiner Bioscience). For cell
97
detachment Trypsin (Sigma Aldrich) was used. All the cells were maintained in
98
culture for 15 passages as maximum to maintain cell identity. Cells were routinely
99
tested for mycoplasma via PCR. The reagents were dissolved in Dimethyl sulfoxide
100
(DMSO) in specified concentrations and added to the cells.
101
102
DNA transfection and infection
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For DNA transfection, a mix of 2,5
μ
g plasmid DNA, 200
μ
l free medium and 5
μ
l PEI
104
was added into the 6-well dish medium (60% confluence), after 6 h incubation at
105
37°C the medium was changed to full supplemented medium. For DNA infection
106
retroviruses or lentiviruses (MOI=10) were added to the cell medium in the presence
107
of polybrene (5
μ
g/ml) and incubated at 37°C for 72 h. After incubation, infected cells
108
were selected with 2
μ
g/ml puromycin for 72 h or 20 µg/ml blasticidin for 10 days.
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110
X-ray irradiation
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Irradiation was performed at room temperature using a 6 MV Siemens linear
112
accelerator (Siemens, Concord, CA) at a dose rate of 9,5 Gy/min.
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114
Colony forming
115
Dependent on the experiment cells were treated with two different protocols. With
116
the direct seeding protocol exponential growing cells were seeded to 10 cm dishes in
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adequate amount to be 50-80% confluent next day. Cells were trypsinized, counted
118
and diluted. The dilution was dispensed into different vials and cells were irradiated
119
in suspension. Cells were directly seeded in adequate amounts into 10 cm plates to
120
obtain 100-400 colonies per dish. With the re-seeding protocol exponential growing
121
cells were seeded to 10 cm dishes in adequate amount to be 25-30% confluent next
122
day. The attached cells were treated with different substances or DMSO as a control.
123
3 h after treatment cells were irradiated with 0, 2, 3, 5, 7, 8 Gy and cultured for 24 h,
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then cells were trypsinized, counted and re-seeded in adequate amounts into 10 cm
125
plates to obtain 100-400 colonies per dish. For both protocols KP and KPP cells
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formed colonies after 6 days, BEAS-2B cells formed colonies after 10-11 days. Cells
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were fixed with ice cold 25% acidic acid in methanol and stained with 0,5% crystal
128
violet. Colonies were count manually. Only colonies containing at least 50 cells were
129
scored. Surviving fractions were calculated by dividing the plating efficiency for the
130
specified dose divided by the plating efficiency of untreated cells. Radiation
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treatment survival curves were fitted to the linear-quadratic model formula S= exp[-
132
α
D-
β
D
2
] (S=survival fraction; D=radiation dose;
α
and
β
fitted parameters). Curves
133
were fitted and blotted using a non-linear regression and analysed with
OriginPro
134
.CC-BY 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 25, 2021. ; https://doi.org/10.1101/2021.07.24.453632doi: bioRxiv preprint

4
(OriginPro, 2020, OriginLab Corporation, Northampton, MA, USA)
.
Mean survival
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fractions at 2 Gy (SF2) and 4 Gy (SF4) were also obtained for each cell model and
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each substance and used to calculate the radiation enhancement ratio at 2 Gy
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(RER
2Gy
) and 4 Gy (RER
4Gy
) RER greater than 1 indicates enhancement of
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radiosensitivity, RER below the value of 1 indicates a radio resistance effect.
139
Similarly, the radiation dose with 25% (D
25
) and 50% (D
50
) survival under different
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conditions was calculated to obtain the dose enhancement ratio (DER
25
and DER
50
)
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that is calculated by dividing D
25
without substance treatment by D
25
with substance
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treatment, respectively D
50
without substance treatment by D
50
with substance. DER
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greater than 1 indicates a radio sensitising effect, a DER below the value of 1
144
indicates a radio protecting effect. Plating efficiency was calculated by dividing the
145
number of colonies by the number of seeded cells. All calculated parameters are
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listed in supplementary table 1 (Table S1)
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Immunological methods
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Cells were lysed in RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM
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Na2EDTA, 1 mM EGTA, 1% NP-40 and 1% sodium deoxycholate), containing
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proteinase inhibitor an phosphatase inhibitor (1/100; Bimake) by sonication using
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Branson Sonifier 150 with a duty cycle at 25%, output control set on level 2 and the
153
timer set to 15 s. Protein concentration was quantified using Bradford assay (Biorad).
154
After mixing of Bradford reagent with 2
μ
l of sample, the photometer was used to
155
normalize the protein amounts with a previously performed bovine serum albumin
156
(BSA) standard curve. The quantified protein (40-80
μ
g) was heated in 4x sample
157
buffer (Thermo Fisher) and 10% sample reducing agent (Thermo Fisher) for 10 min
158
at 70°C and separated on 4-12% Bis/Tris-gels or 3-8% Tris/Acetat-Gels (Thermo
159
Fisher). After separation, protein was transferred to nitrocellulose membrane
160
(Thermo Fisher) in transfer buffer (Thermo Fisher) and then, incubated with blocking
161
buffer (5% low fat milk powder in TBS and 0.1% Tween20) for 60 min at RT. After
162
blocking, membranes were incubated with indicated Primary antibodies (1/1000
163
dilution in a buffer composed 5% low fat milk powder or 5% BSA in TBS and 0.1%
164
Tween20) over night at 4°C. Secondary HRP coupled antibody (Dako 1/1000 dilution
165
in a buffer composed 5% low fat milk powder or 5% BSA in TBS and 0.1% Tween20)
166
were incubated for 2 h at 4°C. Membranes were incubated for 5 min in luminol-
167
solution (250 mg luminol in 100 mM Tris pH 8,6) with 10% v/v cumarinic acid solution
168
(1,1 g cumarinic acid in DMS0 and 0,1% v/v H
2
O
2
)at RT, then membranes were
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recorded with my ECL Imaging System. Analysis and quantifications of protein
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expression was performed using Image Studio software (Licor Sciences, Lincoln,
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NE, USA). Antibodies used for this publication are listed in supplemantary table 2
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(Table S2)
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AnnexinV/DAPI staining:
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Cells growing as sub-confluent monolayers were pretreated with substance for 3 h
176
before radiation with 0 Gy and 8 Gy. The cells were kept under standard conditions
177
for normal cell growth. 24 h, 48 h, 72 h and 96 h after radiation cells were harvested
178
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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5
with trysinization.
Non irradiated cells, treated with camptothecin 5 µM (CPT) were
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harvested 48 h after treatment. Supernatant of cell culture dishes was pooled with
180
trypsinized cells and pelleted by centrifugation. Further preparation for FACS
181
measurement was following the protocol of the BioLegend APC Annexin V Apoptosis
182
Detection Kit and DNA-staining with DAPI Reagent (25
μ
g/mL) (Biolegend, San
183
Diego, CA, USA). 20 000 cells were assayed using a flow cytometer FACSCantoII
184
(Becton Dickinson, San Jose, CA, USA). The output data presented as two-
185
dimensional dot plot. Samples were analyzed using the Flowing software gating
186
events to avoid debris, then dividing events in four quadrants. Flowing software was
187
obtained from P. Terho (Turku Centre for Biotechnology, Turku, Finland). Column
188
histograms and statistics were analyzed with Graphpad PRISM 8 (GraphPad
189
Software, San Diego, California USA) and OriginPro. (OriginPro, 2020, OriginLab
190
Corporation, Northampton, MA, USA)
.
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sgRNA design
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sgRNAs were designed using the CRISPRtool (https://zlab.bio/guide-design-
194
resources).
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AAV and lentivirus production and purification
197
Virus was packaged and synthetized in HEK 293T cells seeded in 15 cm-dishes.
198
For AAV production, cells (70% confluence) were transfected with the plasmid of
199
interest (10
μ
g), pHelper (15
μ
g) and pAAV-DJ or pAAV-2/8 (10
μ
g) using PEI
200
(70
μ
g). After 96 h, the cells and medium of 3 dishes were transferred to a 50 ml
201
Falcon tube together with 5 ml chloroform. Then, the mixture was shaken at 37°C for
202
60 min and NaCl (1 M) was added to the mixture. After NaCl is dissolved, the tubes
203
were centrifuged at 20 000 x g at 4°C for 15 min and the chloroform layer was
204
transferred to another Falcon tube together with 10% PEG8000. As soon as the
205
PEG800 is dissolved, the mixture was incubated at 4°C overnight and pelleted at 20
206
000 x g at 4°C for 15 min. The pellet was resuspended in PBS with MgCl2 and
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0.001% pluronic F68, then, the virus was purified using Chloroform and stored at -
208
80C. AAV viruses were titrated using Coomassie staining and RT-PCR using AAV-
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ITR sequence specific primers.
210
For Lentivirus production, HEK 293T cells (70% confluence) were transfected with
211
the plasmid of interest (15
μ
g), pPAX (10
μ
g) and pPMD2 (10
μ
g) using PEI (70
μ
g).
212
After 96h, the medium containing lentivirus was filtered and stored at -80°C.
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In vivo experiments and histology
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All in vivo experiments were approved by the Regierung Unterfranken and the ethics
216
committee under the license numbers 2532-2-362, 2532-2-367, 2532-2-374 and
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2532-2-1003. The mouse strains used for this publication are listed. All animals are
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housed in standard cages in pathogen
‐
free facilities on a 12 h light/dark cycle
219
with ad libitum access to food and water. FELASA2014 guidelines were followed for
220
animal maintenance.
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 25, 2021. ; https://doi.org/10.1101/2021.07.24.453632doi: bioRxiv preprint

Frequently asked questions

Q1. What is the role of a phosphorylated PTEN in cancer cells?

ATM-mediated PTEN phosphorylation promotes PTEN nuclear 1315 translocation and autophagy in response to DNA-damaging agents in cancer cells. 

Q2. What is the % gene that is altered?

%Genetic Alteration Inframe Mutation (putative driver) Missense Mutation (putative driver) Missense Mutation (unknown significance) Truncating Mutation (putative driver) Truncating Mutation (unknown significance) Fusion Amplification (putative driver) Amplification (unknown significance) Deep Deletion (putative driver) Deep Deletion (unknown significance)mRNA High mRNA Low No alterationsProfiled in mRNA expression z-scores relative to diploidsamples (RNA Seq V2 RSEM)Yes NoPTENKRASHRASNRASEGFRBRAFPIK3CA38%26%8%16%13%23%75%Genetic 

Q3. What is the genesis of 1318 murine lung cancer?

Implementation of CRISPR/Cas9 Genome Editing to Generate 1318 Murine Lung Cancer Models That Depict the Mutational Landscape of Human 1319Disease. 

Q4. who is the author/funder of the preprint?

1252.CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.