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Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

Bol GM, Vesuna F, Xie M, Zeng J, Aziz K, Gandhi N, Levine A, Irving A, Korz D, Tantravedi S, Heerma van Voss MR, Gabrielson K, Bordt EA, Polster BM, Cope L, van der Groep P, Kondaskar A, Rudek MA, Hosmane RS, van der Wall E, van Diest PJ, Tran PT, Raman V - EMBO Mol Med (2015)

Bottom Line: We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity.Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells.Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

Effect of RK-33 on radiation-induced DNA damageA Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT–PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.Source data are available online for this figure.
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fig09: Effect of RK-33 on radiation-induced DNA damageA Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT–PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.Source data are available online for this figure.

Mentions: As RK-33 promoted radiation sensitization, we evaluated the DNA damage response following combination treatment with RK-33 and γ-radiation. Following γ-radiation, 53BP1 and γ-H2AX foci numbers increased within an hour and returned to pre-radiation foci numbers by 24 h. However, 53BP1 and γ-H2AX foci persisted 24 h post-RK-33 treatment, indicating reduced or delayed DNA repair (Fig9A). The extent of the impaired DNA damage repair was quantified by counting the number of severely damaged cells (> 10 foci per nucleus). This was determined for both 53BP1 foci (Fig9A, top panel) and γ-H2AX foci (Fig9A, bottom panel). Most cells were severely damaged 1 h after radiation, but a large reduction in 53BP1 and γ-H2AX foci after 6 h and normalization after 24 h, without significant cell death, indicated proficient DNA damage repair in untreated A549 cells. Importantly, when these cells were pre-treated with RK-33, DNA damage persisted well beyond 24 h (Fig9B).


Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

Bol GM, Vesuna F, Xie M, Zeng J, Aziz K, Gandhi N, Levine A, Irving A, Korz D, Tantravedi S, Heerma van Voss MR, Gabrielson K, Bordt EA, Polster BM, Cope L, van der Groep P, Kondaskar A, Rudek MA, Hosmane RS, van der Wall E, van Diest PJ, Tran PT, Raman V - EMBO Mol Med (2015)

Effect of RK-33 on radiation-induced DNA damageA Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT–PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.Source data are available online for this figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492822&req=5

fig09: Effect of RK-33 on radiation-induced DNA damageA Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT–PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.Source data are available online for this figure.
Mentions: As RK-33 promoted radiation sensitization, we evaluated the DNA damage response following combination treatment with RK-33 and γ-radiation. Following γ-radiation, 53BP1 and γ-H2AX foci numbers increased within an hour and returned to pre-radiation foci numbers by 24 h. However, 53BP1 and γ-H2AX foci persisted 24 h post-RK-33 treatment, indicating reduced or delayed DNA repair (Fig9A). The extent of the impaired DNA damage repair was quantified by counting the number of severely damaged cells (> 10 foci per nucleus). This was determined for both 53BP1 foci (Fig9A, top panel) and γ-H2AX foci (Fig9A, bottom panel). Most cells were severely damaged 1 h after radiation, but a large reduction in 53BP1 and γ-H2AX foci after 6 h and normalization after 24 h, without significant cell death, indicated proficient DNA damage repair in untreated A549 cells. Importantly, when these cells were pre-treated with RK-33, DNA damage persisted well beyond 24 h (Fig9B).

Bottom Line: We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity.Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells.Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus