Vinculin
Natthakan Thongon Department of Leukemia Colla’s Lab
Targeting DNA2 Overcomes Metabolic Reprogramming in 1q21 Multiple Myeloma
ILF2
g H2AX
MM cells can overcome ILF2 ASO-induced DNA damage. Cleaved caspase 3
Antisense therapy targeting ILF2 (ILF2 ASOs) induces DNA damage in 1q21 MM cells.
NT ASOs
ILF2 ASOs
1 2 3 1 2 3 ILF2 ASOs NT ASOs
NT ASOs
ILF2 ASOs
NT ASOs
ILF2 ASOs
! H2AX DAPI
JJN3
JJN3
KMS11
1 2 3 1 2 3
40
NT ASOs ILF2 ASOs
Vinculin
1 2 3 1 2 3
1 2 3 1 2 3
✱✱
NT ASOs
ILF2 ASOs 3 weeks
30
Vinculin
ILF2
✱✱
20
ILF2
g H2AX
ILF2 ASOs
10
Cleaved caspase 3
g H2AX
0
Cleaved caspase 3
KMS11
JJN3
JJN3
JJN3
KMS11
KMS11
1 week
3 weeks
Western blot (left), immunofluorescence (middle), and apoptosis (right) analyses in KMS11 and JJN3 cells treated with non-targeting (NT) or ILF2 ASOs for 1 week.
Aim #1: To dissect the molecular mechanisms by which MM cells overcome ILF2 ASO-induced DNA damage • Resistance to ILF2 ASOs is not induced by clonal selection (scRNA-seq) • ILF2 ASO-resistant MM cells undergo metabolic switch and are dependent on oxidative phosphorylation to maintain survival (scRNA-seq and metabolomic analysis)
OXPHOS mediates MM resistance to ILF2 ASOs.
75
IACS-010759 Veh
✱✱✱✱
✱✱✱✱
Cluster 1
NT ILF2
ILF2
MYC TARGETS V1 OXIDATIVE PHOSPHORYLATION MTORC1 SIGNALING UNFOLDED PROTEIN RESPONSE
50
✱✱✱
UV RESPONSE UP DNA REPAIR
25
ILF2 ASO-treated JJN3 for 1 wk or for 3 wks were further treated with the OXPHOS inhibitor IACS-010759 for 72 hours.
P value <10 -4
✱✱
G2M CHECKPOINT PI3K AKT MTOR SIGNALING PROTEIN SECRETION REACTIVE OXYGEN SPECIES PATHWAY
<10 -22 <10 -10 <10 -6 <10 -20
0
Cluster 2
0
5
10
15
20
-Log 10 (p-value)
Single cell RNA-seq and metabolomic analysis were performed in JJN3 treated with NT or ILF2 ASOs for 3 weeks.
Natthakan Thongon Department of Leukemia Colla’s Lab
Targeting DNA2 Overcomes Metabolic Reprogramming in 1q21 Multiple Myeloma
CRISPR/Cas9-based screening to identify DNA repair effectors whose loss of function suppresses MM cells’ resistance to ILF2 ASO-induced DNA damage
Targeting DNA2 enhances ILF2 ASO-induced apoptosis in JJN3 cells.
NT ASOs
ILF2 ASOs Veh NSC
90
Veh NSC
2
✱✱✱
✱✱✱✱
Vinculin
60
✱✱✱
0
ILF2 Cleaved caspase 3
30
DNA2 DNA2
-2
MMS19 DDB1 PRPF19 DNA2
g -H2AX
0
0
50
100
150
200
Rank
Western blot (left) and apoptosis (right) analyses were performed in JJN3 cells treated with vehicle (Veh) or the DNA2 inhibitor NSC105808 (NSC; 1µM) for 48 hours after long-term exposure to NT or ILF2 ASOs.
sgRNAs targeting MMS19 , DNA2 , and DDB1 genes were significantly depleted in ILF2 ASO-treated JJN3 cells but not KMS11 cells after 3 weeks of ASO-treatment. DNA2 is the only druggable target.
Aim #2: To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality in MM cells undergoing metabolic reprogramming in the context of ILF2 depletion DNA2 inhibition decreases the oxygen consumption rate and increases ROS production in ILF2-depleted cells.
75
NT ASOs NT ASOs + NSC ILF2 ASOs ILF2 ASOs + NSC
500
✱✱✱
Oligomycin FCCP R/A
✱✱✱
60
400
45
300
✱✱✱
30
✱✱✱
200
Seahorse experiments (left) were performed in JJN3 cells treated with ASOs for 7 days prior to receiving NSC for 72 hours. Quantification of ROS production (middle) and transmission electron microscopy (right) were performed in JJN3 cells treated with ASOs for 7 days prior to receiving NSC for 48 hours.
15
100
0
0
0
20
40
60
80
Time (minutes)
Natthakan Thongon Department of Leukemia Colla’s Lab
Targeting DNA2 Overcomes Metabolic Reprogramming in 1q21 Multiple Myeloma
Working model & Conclusion
ü DNA2 is essential to maintain oxidative phosphorylation and metabolic reprograming in MM cells. ü DNA2 inhibition is a synthetic lethal approach to targeting 1q21 MM cells in the setting of ILF2 depletion-induced DNA damage. • To evaluate whether inhibition of DNA2 activity is synthetically lethal in MM plasma cells from patients whose disease failed previous therapies, such as therapy with proteasome inhibitors. Future direction
??
↑↑ OXPHOS
Contact information Presenting author: nthongon@mdanderson.org Corresponding author: scolla@mdanderson.org Department of Leukemia, MD Anderson Cancer Center, Houston, TX, 77054
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