Targeting DNA2 Overcomes Metabolic Reprogramming in Multipl…

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

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↑↑ 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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