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. 2024 Jan 16;5(1):101354.
doi: 10.1016/j.xcrm.2023.101354. Epub 2024 Jan 5.

Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

Shipra Malik  1 , Sai Pallavi Pradeep  1 , Vikas Kumar  1 , Yong Xiao  2 , Yanxiang Deng  3 , Rong Fan  4 , Juan C Vasquez  5 , Vijender Singh  6 , Raman Bahal  7
Affiliations

Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

Shipra Malik et al. Cell Rep Med. .

Abstract

Targeting oncogenes at the genomic DNA level can open new avenues for precision medicine. Significant efforts are ongoing to target oncogenes using RNA-targeted and protein-targeted platforms, but no progress has been made to target genomic DNA for cancer therapy. Here, we introduce a gamma peptide nucleic acid (γPNA)-based genomic DNA-targeted platform to silence oncogenes in vivo. γPNAs efficiently invade the mixed sequences of genomic DNA with high affinity and specificity. As a proof of concept, we establish that γPNA can inhibit c-Myc transcription in multiple cell lines. We evaluate the in vivo efficacy and safety of genomic DNA targeting in three pre-clinical models. We also establish that anti-transcription γPNA in combination with histone deacetylase inhibitors and chemotherapeutic drugs results in robust antitumor activity in cell-line- and patient-derived xenografts. Overall, this strategy offers a unique therapeutic platform to target genomic DNA to inhibit oncogenes for cancer therapy.

Keywords: HDACi; PNA; c-Myc; cancer therapy; chemotherapy; histone deacetylase inhibitors; lymphoma; nuclear delivery; peptide nucleic acid; transcription inhibition.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Design of anti-transcription γPNA-NLS to target the c-Myc oncogene (A) Schematic of γPNA-NLS-mediated inhibition of c-Myc transcription. (B) Graphic representation of target sites in P1 and P2 of the human c-Myc oncogene. (C) Design of gamma peptide nucleic acid (γPNA) conjugated with an NLS to target the indicated sites. (D) PAGE assay of γPNA1-NLS and γPNA2-NLS at increasing PNA concentrations with dsDNA 1 and 2 containing the respective target site. Scr-γPNA3-NLS was incubated with dsDNA2. (E) Flow cytometry histograms of lymphoma cells after 24 h treatment with the indicated concentrations of γPNA5-NLS-T. (F) Confocal microscopy images of live lymphoma cells after 24 h treatment with γPNA5-NLS-T (8 μM). Scale bars, 15 μm. (G) Amplicon assay confirming binding of γPNA2-NLS (8 μM) to target site 2 in lymphoma cells after 24 h treatment.
Figure 2
Figure 2
Anti-transcription activity of γPNA-NLS in lymphoma cells (A–C) Relative fold change in c-Myc and EZH2 after treatment with PBS and the indicated γPNAs (8 μM) for 24 h. (D–F) Percentage of viability of lymphoma cells treated with PBS, γPNA1-NLS, γPNA2-NLS, and Scr-γPNA3-NLS for 24 h. (G and H) Fold change in live and early apoptotic lymphoma cells after 24 h treatment with γPNA2-NLS and Scr-γPNA3-NLS (8 μM). (I) Relative change in c-Myc in U2932 cells treated with the indicated γPNA2-NLS doses in comparison with Scr-γPNA3-NLS. (J and K) Representative WBs of c-MYC protein in lymphoma cells (top) after 24 h treatment with PBS (control), γPNA2-NLS, and Scr-γPNA3-NLS (8 μM). Graphs represent the quantification of c-MYC protein fold change relative to PBS. (A–K) Graphs show mean ± SEM (n = 3); p value for unpaired two-sample t test. (L) WB representing levels of γH2AX protein in γPNA2-NLS and Scr-γPNA3-NLS (8 μM) after 24 h relative to PBS. (M) Representative immunofluorescence images of HeLa cells stained with γH2AX antibody after 24 h treatment with γPNA2-NLS (8 μM) and bleomycin. Scale bar, 50 μm.
Figure 3
Figure 3
Transcriptome sequencing analysis after c-Myc silencing (A) GO analysis of downregulated DEGs. (B) RT-PCR-based validation of the downregulated DEGs associated with DNA replication and repair in U2932 cells treated with PBS (control), γPNA2-NLS, and Scr-γPNA3-NLS (8 μM) for 72 h. Graphs show mean ± SEM (n = 3); p value for unpaired two-sample t test. (C) Hierarchical clustering analysis of DEGs after treatment of U2932 cells with γPNA2-NLS and siRNA compared with PBS treatment. (D) Enrichment plots for the top three datasets enriched in GSEA, showing NES and normalized p value.
Figure 4
Figure 4
Efficacy of anti-transcription γPNA-NLS in lymphoma xenograft mice (A) IVIS images of U2932 xenografts post γPNA5-NLS-T administration at the indicated time points (5 mg/kg dose, i.v.). (B) IVIS images of tumor and organs, including kidneys (K), lungs (Lu), liver (L), spleen (S), intestine (In), and bone marrow (BM), after γPNA5-NLS-T administration. (C) Confocal images of γPNA5-NLS-T treated xenografts. Blue, DAPI. (D and E) c-MYC and EZH2 protein levels (D) and quantification (E) in control and γPNA2-NLS-treated (25 mg/kg, i.v.) U2932 xenografts after 24 h (control, n = 6; γPNA2-NLS, n = 7). (F and G) c-MYC and EZH2 protein levels (F) and quantification (G) in control and γPNA2-NLS-treated (5 mg/kg, i.t.) Raji xenografts after 24 h (control, n = 5; γPNA2-NLS, n = 6). (E and G) Graphs show mean ± SEM; p value for unpaired two-sample t test. (H) Tumor growth curve of U2932 xenograft mice treated with γPNA2-NLS and Scr-γPNA3-NLS (5 mg/kg) (saline, n = 10; γPNA2-NLS, n = 9; Scr-γPNA3-NLS, n = 8). Mean ± SEM; p value for two-way ANOVA. (I) Immunohistochemistry of tumors, including Ki67 and caspase-3, staining in U2932 xenografts post survival study. Scale bar, 50 μm. (J) Serum chemistry analysis of U2932 xenografts post survival. Graphs show mean ± SEM (n = 3).
Figure 5
Figure 5
Efficacy of γPNA2-NLS in combination with HDACis and chemotherapeutic drugs (A) Percentage viability of U2932 cells treated with the indicated γPNA-NLS, HDAC inhibitors, and combination at 1, 2, and 4 days. (B) Fold change of c-Myc and EZH2 gene expression in treated U2932 cells on day 4. (C) WB analysis representing c-MYC and EZH2 protein levels in U2932 cells on day 4. (D) Fold change in c-Myc and EZH2 levels in Raji cells after 24 h. (E) Percentage of viability of U2932 cells treated with doses of CHOP or in combination with γPNA2-NLS after 24 h. (A, B, D, and E) Graphs show mean ± SEM (n = 3); p value for unpaired two-sample t test. (F) Workflow representing the in vivo study plan to evaluate the efficacy of γPNA2-NLS with romidepsin (top) and tumor growth curve of U2932 xenografts after treatment with romidepsin and combination of romidepsin with γPNA2-NLS (bottom). Vehicle, n = 5; romidepsin, n = 8; γPNA2-NLS+romidepsin, n = 8. (G) Representative immunohistochemistry images of H&E, Ki67, and caspase-3 staining in U2932 xenografts post survival. (F and G) Graphs show mean ± SEM (n = 3); p value for two-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.0001. (H) Workflow representing the in vivo study plan to evaluate γPNA2-NLS efficacy with CHOP (top) and tumor growth curve of U2932 xenografts treated with CHOP and γPNA2-NLS+CHOP (bottom). Control, n = 6; CHOP, n = 7; γPNA2-NLS+CHOP, n = 6. (I) Representative immunohistochemistry images of H&E, Ki67, and caspase-3 staining in control, CHOP-, and γPNA2-NLS+CHOP-treated U2932 tumors post survival. (G and I) Scale bars, 100 μm (H&E) and 50 μm (immunohistochemistry).
Figure 6
Figure 6
Anti-transcription efficacy of γPNA-NLS in Eμ-myc transgenic mice (A) In vivo biodistribution of γPNA-NLS-T (5 mg/kg) in the LNs and organs of Eμ-myc mice. (B) Confocal images after 24 h of γPNA-NLS-T i.v. and s.c. administration. (C) Confocal images of LNs isolated from control and γPNA-NLS-T-treated (5 mg/kg) mice after 6 h. (B and C) Scale bars 30 μm. (D) Workflow representing the study plan for evaluating γPNA4-NLS efficacy in Eμ-myc mice (top), representative images of the LNs from control and γPNA4-NLS-treated mice (bottom left), and graph representing the mass of LNs from control and γPNA4-NLS post treatment (bottom right). (E and F) c-MYC protein levels in Eμ-myc mice on day 3 (left). The graph represents the protein quantification (right). (G) Representative immunohistochemistry images from control and γPNA4-NLS-treated mice. Scale bar, 50 μm. (H) Relative fold change in the level of cytokines in γPNA4-NLS-treated Eμ-myc mice on day 3 relative to the control group. Graph shows mean ± SEM (n = 3); p value for unpaired two-sample t test.
Figure 7
Figure 7
Efficacy of γPNA2-NLS in the DLBCL patient-derived xenograft (PDX) mouse model (A) Tumor growth curve of DLBCL PDX mice treated with γPNA-NLS, HDACis, and combination (control, n = 4; γPNA2-NLS, n = 6; romidepsin, n = 6; γPNA2-NLS+romidepsin, n = 6). (B) Graph representing tumor volumes of the indicated groups. Control, n = 4; γPNA2-NLS, n = 5; romidepsin, n = 5; γPNA2-NLS+romidepsin, n = 5. (C) Representative images of mice bearing tumors from the indicated treatment groups. (D) Representative immunohistochemistry images from different groups. Scale bar, 50 μm. (E) Boxplot representing quantification of Ki67 in tumor sections. (F) Tumor growth curve of mice treated with CHOP and combination of γPNA2-NLS with CHOP (control, n = 5; CHOP, n = 6; γPNA2-NLS+CHOP, n = 5). (G) Graph representing the tumor volumes from the indicated treatment groups (control, n = 4; CHOP, n = 5; γPNA2-NLS+CHOP, n = 5). (H) Representative images of mice bearing tumors from the indicated groups. (I) Representative images of H&E- and Ki67-stained tumor sections Scale bar, 50 μm. (J) Boxplot representing Ki67 quantification in tumor sections (control, n = 2; CHOP, n = 2; γPNA2-NLS+CHOP, n = 3). Results are presented as mean ± SEM; p value for unpaired two-sample t test. For tumor growth curves: p value for two-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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