We have previously described the development of the c-MYC-mRNA drug (Awah et al., 2024) and its mechanism of action. We discovered the stable mRNA poly U sequences on the 3′UTR of c-MYC and engineered them to unstable forms under the control of mRNA de-capping promoter DCP1A. The destabilized mRNA drug directly binds to its target mRNA recognition site either in-frame or at the 3′UTR or both and triggers the stalling of the ribosome. This stalling is sensed by the PELO-EXOSC4-RPL3 complex, which triggers the degradation of the target transcript. This process does not affect normal healthy cells because the healthy cells do not differentially express EXOSC4, PELO, or RPL3.

We obtained the following pancreatic cancer cells: PSN1 (RRID: CVCL_1644), MIA-Paca2 (RRID: CVCL_0428), PANC-1 (RRID: CVCL_0480), AC16 (RRID: CVCL_4U18), and RWPE1 (RRID: CVCL_3791) from ATCC. The PSN1 was grown in RPMI media supplemented with antibiotics/antimycotics before use. The PANC-1 and MIA-Paca-2 were grown in DMEM supplemented with antibiotics and anti-mycotics before use. FBS (10%) was added to the media. AC16 and RWPE1 were grown using the standard media purchased from ATCC. The cells were authenticated by ATCC by short-term repeat (STR) sequencing, and we regularly evaluated them for mycoplasma before use.

We determined the IC50 of the MYC-mRNA drug 3′UTRMYC1-18 by titrating the drug in a dose-dependent manner in a head-to-head comparison with the standard-of-care drugs (olaparib, paclitaxel, cisplatin, actinomycin D, bevacizumab, cyclophosphamide, MYCi975 (MYC-Max inhibitor), and osimertinib. All drugs were obtained from Selleckchem, USA. The doses used are from 2.5 µg to 40 µg. Serial dilutions of the drugs were made. We seeded the cells at 5,000 cells per well in a 96-well plate. The cells were allowed to attach for 24 h, and then the drugs were added. We incubated the treated cells for 72 h. We read the viability using the cell titre glo (Promega G7570) and normalized it to the controls. The data curves were fitted on the drug dose–response chart in GraphPad Prism (USA), and the IC50 values were derived.

To quantify the dose-dependent downregulation of c-MYC transcript in pancreatic cancer, we treated the MIA-Paca-2 cells with a dose-dependent concentration of 3′UTRMYC1-18. We extracted the RNA using the Qiagen RNeasy kit (Cat No. 74104). The RNA was stored at −80°C before use. To reverse transcribe the RNA, we used the Superscript IV reverse transcriptase kit (Cat No. 18090200) to make cDNA. We designed the qPCR primers targeting the exons of MYC and GAPDH as a housekeeping gene control. All the primers used are described in ref 13. The delta CT was used to normalize the transcript expression.

The iron oxide nanocage and the MYC-mRNA drugs were complexed in a 1:1 ratio (Awah et al., 2024; Rampersaud and Fang, 2016), and the conjugates were incubated overnight before use at 4°C. Subsequently, the drug conjugate was found to be stable at room temperature, 4°C, and −20°C till use.

We performed animal studies to validate the in vivo therapeutic efficacy of the c-MYC-mRNA drug in lethal pancreatic cancer. We obtained Institutional Animal Care and Use Committee (IACUC) institutional approval from the CUNY Institutional Review Board. We ordered five female and three male NSG mice aged 5–8 weeks and weighing 15–21 g, from the Jackson Laboratory. Once received, we allowed the mice to acclimatize according to the protocol. We implanted 10 million PSN1 cells into the flanks of the mice. Seven days post implantation, the tumor engrafted and, on day 10, we randomized the mice into a vector + nanocage group (N = 4) and a 3× IC50 3′UTRMYC1-18 + nanocage group (N = 4). All mice had tumors, and there were no excluded mice. We started treating the mice intravenously with the 3XIC50 dose of the drug, which is 3.5 µg (as determined from Figure 1), 2× per week. On day 25, we collected the blood of the mice to assess for the safety profile (full blood count, electrolyte, liver, kidney, pancreatic, and gall bladder function). The vector + nanocage-treated tumor-bearing mice died on day 26 at a pre-determined tumor volume of 0.8 cm³, upon which the animal was euthanized in 100% CO₂. The 3× IC50 group lived till day 32.

To establish the dose-dependent inhibition of tumor volume by the MYC-mRNA drug in pancreatic cancer, we obtained eight mice (seven females, one male) from the Jackson Laboratory, age 5–8 weeks and weighing 15–20 g. We allowed the mice to acclimatize and implanted 10 million PSN1 cells into their flanks. By day 7, they were engrafted, and we randomized them into three groups based on tumor size and weight. The groups were 1) vector + nanocage (N = 2); 2) 6× IC50 21 µg (N = 3); 3) 9× IC50 31 µg (N = 3). We then dosed the mice 2× per week intravenously through the tail veins till day 28. By day 27, the vector + nanocage-treated mice had died. The 6× IC50-treated group died on day 33. We dosed the 9× IC50 1× per week from day 28 to day 33, after which we stopped dosing. The mice in the 9× IC50 group lived till day 55 and were euthanized. We recorded daily tumor volumes, weights, and body condition scores. The mice were euthanized with CO₂ at the end of the study. The log-rank Mantel test was used to determine statistical significance between the in vivo treatment groups. The Kaplan–Meier survival curve was used to determine survival differences between the various treatment groups and controls. Mice were weighed daily; body condition scores and tumor volume were measured with calipers as 1/2×L×W×W and recorded and documented.

To determine the safety of the c-MYC-mRNA drugs to the blood cells, serum electrolytes, kidney, liver, pancreatic, and gall bladder function were monitored. We collected blood and serum and sent them to the Memorial Sloan Kettering Cancer Core Pathology laboratory, which ran the full blood count, lipid profile, liver enzymes, kidney function, and electrolyte analyses. The reference is normal NSG mice. The analyst was blinded to the experimental details.

The MYC-mRNA drug is complexed with the iron oxide nanocage. To understand the pharmacokinetics of the drug in vivo in the tumor-bearing mice, we administered 3′UTRMYC1-18 + IO nanocage (8.7 µg) intravenously; we collected the blood at 0 h, 3 h, 6 h, and 72 h and then obtained the serum. For each time point, we collected serum and then detected the iron oxide using an ELISA-based assay (Catalog #: HT20, Sigma Aldrich). We measured the absorbance intensity at various time points and presented the results. Our results demonstrate that 3′UTRMYC1-18 has a relatively stable half-life in the serum of greater than 24 h. The measurement of the iron oxide nanocage is a surrogate marker to easily determine the pharmacokinetics of 3′UTRMYC1-18 complexed with the iron oxide nanocage.

We previously described a Prussian blue stain approach that identified iron oxide nanocages in the tumors, livers, lungs, and brains of the tumor-bearing mice treated with the MYC-mRNA drug IO-nanocage complex in Awah et al. (2024).

Once the mice died, we took the fresh carcasses, dissected them, and collected the tumors, lungs, kidneys, livers, and brains. We rinsed the lungs in 1X PBS and took the images of the fresh organs. Subsequently, we collected fresh tissues of tumors and organs and froze them at −80°C. The tissues were then placed in 4% paraformaldehyde till they were sent to the pathology lab for paraffin embedding and hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining.

The H&E images of the vector + nanocage (exp #1) and 3× IC50 groups were performed by the core pathology group of the Memorial Sloan Kettering Cancer Center. The H&E samples from the vector + nanocage (experiment #2), 6× IC50, and 9× IC50 groups were sectioned by QC and stained by JPD. The images of the stained tissues were obtained using an EVOS FL microscope at ×40 magnification, and quantification was done in ImageJ (RRID:SCR_003070) (https://imagej.net/ij/) and visualized in GraphPad Prism (v10) RRID:SCR_002798.

Briefly, to perform IHC staining against MYC and PD-L1 from the tumors and organs of the treated mice and the controls, we used the Abcam IHC protocol (https://www.abcam.com/en-us/technical-resources/protocols/ihc-with-samples-in-paraffin). We deparaffinized the tissue slides according to the protocol and performed enzymatic antigen retrieval using 1:1 trypsin concentrates and buffer. Washes were done with 1X TBST. Blocking was done with protein block (ab64212) for 1 h, after which the washes were done again. Next, we incubated the slides with primary antibodies against MYC (Anti-c-MYC 1:1000, cat no: 67,447-1-lg) or with PD-L1 (E1L3N^® XP^® Rabbit mAb #13684) overnight. Next day, the slides were washed with 1X TBST. The secondary antibody was added to the slide and incubated for 1 h. Subsequently, we washed the slides with 1x TBST and, to detect the signals, we used the DAB concentrate and enhanced the signal with enhancer. We counter-stained, mounting media was added, and sealed the slide with the cover slip . Images were obtained on an EVOS Fl at ×40. Target stains of the MYC and PD-L1 were quantified in ImageJ (https://imagej.net/ij/) RRID:SCR_003070.

The drug dose–response curve experiments to determine the c-MYC-mRNA downregulation by 3′UTRMYC1-18 were performed in multiple replicates and in a minimum number of N = 3. The in vivo dose-dependency experiment was performed in replicate with a minimum of four mice per group to validate therapeutic efficacy in the first experiment. In the second experiment, we used N = 3 mice per group, with an equal distribution of sexes (male and female and age and weight). A paired T-test was used to determine statistical significance between treated groups and controls. The Log Rank Mantel test was used to determine the statistical significance of survival differences between the in vivo treatment groups. The Kaplan–Meier survival curve was used to determine survival differences between the various treatment groups and controls. All data were plotted with GraphPad Prism (v.10) RRID:SCR_002798.

To validate the in vitro therapeutic efficacy of this novel c-MYC-mRNA drug (3′UTRMYC1-18), the metastatic drug-resistant pancreatic cancer cell lines MIA-PaCa-2, PSN1, and PANC1 were used. We performed a head-to-head, dose-dependent IC50 determination with 3′UTRMYC1-18 and the standard-of-care drugs, including a MYC-Max inhibitor (MYCi975). Figures 1A–C show that 3′UTRMYC1-18 achieved an IC50 of 1.98 µM and 1.2 µM, which is superior to these standard-of-care drugs and to MYCi975 in PSN1 and MIA-Paca2 but not in the PANC1 cells. The MYC-mRNA drug engaged the MYC-mRNA in a dose-dependent manner (Figure 1D), and we determined that the drug synergizes with epirubicin in a rational combination (Figure 1E).

To prove the safety of the engineered 3′UTRMYC1-18 to the normal healthy cells of the body, we performed a dose-dependent titration of the drug (3′UTRMYC1-18) in a head-to-head comparison with the standard-of-care drugs and MYCi975 in the normal cardiomyocytes, the AC16 cells, and the normal healthy epithelial cells, the RWPE1 (Supplementary Figures 1A, B). We found that 3′UTRMYC1-18 was not toxic to the normal cardiomyocytes and epithelial cells when compared to the standard-of-care drugs and MYCi975, which were toxic to the healthy cells (Supplementary Figures 1A, B). At the mRNA level, we show that 3′UTRMYC1-18 does not downregulate the MYC expression in healthy AC16 and RWPE1 cells compared to the controls (Supplementary Figures 1C, D).

To prove the in vivo efficacy of the drug, we performed two independent experiments (Figure 2A), both titrating the drug in a dose-dependent manner. The tumor-bearing mice treated with the c-MYC drugs (3.6 µg) show a significant reduction in the tumor volume (*p = 0.019) compared to the controls (Figures 2B,C), with a tumor volume reduction of approximately half (Figure 2B). They also survived better than the vector + nanocage-treated group (*p = 0.0117) (Figure 2C).

To prove that the c-MYC-mRNA drug inhibited c-MYC in vivo in a titratable dose-dependent manner in lethal pancreatic cancer, we titrated the dose of the MYC-mRNA drug from the first in vivo experiment of 3× IC50 (3.6 µg) to 6× IC50 (21.6 µg) and 9× IC50 (32 µg). The control was the vector + nanocage-treated group (Figure 2B). The 9× IC50 group achieved complete in vivo inhibition of pancreatic cancer (Figures 2B,D), with an exceptionally significant survival outcome (Figure 2C), ****P < 0.0001.

To confirm that the MYC-mRNA drug achieved on-target inhibition of c-MYC in the primary tumor to achieve therapeutic efficacy, we performed H&E staining of the tumors (3) from the vector + nanocage, the 3× IC50, 6× IC50, and 9× IC50-treated tumor-bearing mice. We found that the vector + nanocage-treated tumors were hyperchromatic, malignant pleomorphic cells (Figures 3A–C). The 3× IC50 dose achieved complete pathological response in 1/3 and partial response in 2/3 of the tumors (Figures 3D–F). The 6× IC50 dose achieved complete pathological response in 1/3, partial response in 1/3, and no response in 1/3 of the tumors (Figures 3G–I). The 9× IC50 dose achieved 100% complete pathological response (3/3) (Figures 3J–L), quantified in Figures 3M,N. Next, we stained for c-MYC expression by IHC in three tumors from each group. We found a very high level of c-MYC in the vector + nanocage-treated group (Figures 3A–C), a moderate level of c-MYC expression in the 3× IC50-treated tumor group (Figures 3D–F), and complete inhibition of the c-MYC expression in the 6× IC50 and 9× IC50 3′UTRMYC1-18-treated groups (Figures 3G–J,L), quantified in Figure 3O. We found similar dose-dependent downregulation for PD-L1 expression in the same tumors with dose-dependent downregulation of the c-MYC expression (Figures 3A–L), quantified in Figure 3P.

Taken together, these data demonstrate that the c-MYC-mRNA drug achieved dose-dependent, on-target inhibition of the lethal pancreatic tumors via dose-dependent downregulation of the c-MYC and PD-L1 expression.

The liver is the most common site of pancreatic cancer metastasis. We investigated whether inhibition of the tumor and downregulation of the c-MYC and PD-L1 expression led to the inhibition of metastasis to the lungs, liver, and pancreas. We performed H&E staining of the three livers from the vector + nanocage, 3× IC50, 6× IC50, and 9× IC50 groups of the 3′UTRMYC1-18-treated tumor-bearing mice (Figure 4). We found hyperchromatic pleomorphic cells (3/3), perforated (3/3), and eosinophilic hemorrhagic lesions (2/3) in the livers of the vector + nanocage-treated tumor-bearing mice (Figures 4A–C). The 3× IC50 3′UTRMYC-18 dose shows a lesser level of hyperchromatic pleomorphic cells (3/3), few perforations (3/3), and no eosinophilic hemorrhagic lesions (0/3) in the livers of the treated tumor-bearing mice (Figures 4D–F). The 6× IC50 3′UTRMYC1-18 dose achieved more than 75% inhibition of the hyperchromatic pleomorphic cells (3/3), no perforations (0/3), and no eosinophilic hemorrhagic lesions (0/3) in the livers of the tumor-bearing mice treated (Figures 4G–I). The 9× IC50 3′UTRMYC1-18 dose achieved (1/3) inhibition of the hyperchromatic pleomorphic cells, (1/3) perforation, and hemorrhagic lesion in the liver of the tumor-bearing mice (Figures 4J–L). The quantification of the hyperchromatic pleomorphic cells, the liver architecture preservation, and hemorrhagic lesions are shown in Figures 4M,N. We stained for the c-MYC and PD-L1 expression in the control and different treatment groups. We found markedly elevated levels of c-MYC (2/3) and PD-L1 (3/3) in the livers treated with the vector + nanocage (Figures 4A–C) and moderate expression levels of c-MYC and PD-L1 in the 3× IC50 dose-treated group (Figures 4D–F). In the 6× IC50 and 9× IC50 groups, there is complete downregulation of the c-MYC in the liver (Figures 4G–L). We found only (1/3) liver in the 6× IC50 and 9× IC50 with a markedly elevated PD-L1 expression (Figures 4G–L), quantified in Figures 4O,P. Taken together, we show the dose-dependent inhibition of liver metastasis through the downregulation of the c-MYC and PD-L1.

Next, we investigated the lungs to understand whether 3′UTRMYC1-18 inhibited pancreatic cancer metastasis to the lungs. We performed H&E staining of the lungs of the tumor-bearing mice in the vector + nanocage, 3× IC50, 6× IC50, and 9× IC50 3′UTRMYC1-18 groups. We found hyperchromatic, pleomorphic cells (3/3), and eosinophilic hemorrhagic lesions (3/3) in the lungs treated with the vector + nanocage (Figures 5A–C). In the 3× IC50 3′UTRMYC1-18-treated mice, we found malignant hyperchromatic pleomorphic cells (3/3) and (1/3) eosinophilic hemorrhagic lesions in the lungs (Figures 5D–F). In the 6× IC50 and 9× IC50 3′UTRMYC1-18-treated lungs, we found complete inhibition (3/3) of the malignant pleomorphic cells and no eosinophilic hemorrhagic cells in the lungs (Figures 5G–L). We quantified these data in Figures 5M,N.

We extended our analysis into the c-MYC and PD-L1 expression downregulation by comparing the expression of the c-MYC and PD-L1 in the lungs of the vector + nanocage-treated tumor-bearing mice and the dose-dependent treatment groups (Figures 5A–C). We found a clear dose-dependent downregulation of the c-MYC and PD-L1 in the lungs treated with 3×, 6×, and 9× IC50 3′UTRMYC1-18 (Figures 5D–K). The data are quantified in Figures 5O,P. Taken together, we demonstrate that the drug achieved distant organ lung metastasis inhibition by downregulating the c-MYC and PD-L1 in a dose-dependent manner.

In the brain cells, we found the inhibition of brain metastasis in the pancreatic cancer tumor-bearing mice in the 6× and the 9× IC50 3′UTRMYC1-18 groups (Supplementary Figures 2C, D) compared to the controls, the vector + nanocage, and the 3× IC50 3′UTRMYC1-18 groups (Supplementary Figures 2A, B). The inhibition of brain metastasis is dose dependent in the order 9× IC50 = 6× IC50 > 3× IC50 3′UTRMYC1-18 > vector + nanocage-treated groups (Supplementary Figures 2A-D), quantified in Supplementary Figure S2E.

To prove the MYC-mRNA drug safety in the tumor-bearing mice compared to healthy controls, we collected blood on day 25 (Figure 1A) to assay the blood cells, electrolytes, liver enzymes, kidney function, and pancreatic and biliary function analysis from the 3× IC50 group (Supplementary Table S1, Supplementary Table S2). We found no change in red blood cells (Figure 6A), no change in hemoglobulin (Figure 6B) and other blood indices (see Supplementary Table S1), no change in the levels of the blood urea nitrogen levels (Figure 6C), and no change in the weight of the mice that received the dose-dependent mRNA drugs (Supplementary Figure S3A). We found a normal range of creatinine levels (Figure 6D). We examined the liver ALP levels (Figure 7A) and other liver enzyme levels (Supplementary Table S2). We found that all levels were reduced except for the AST levels, which were elevated in one of the tumor-bearing mice treated with 3× IC50 3′UTRMYC1-18 (Supplementary Table S2). The total protein, albumin, and globulin levels were within the same range (Figures 7B–D). The glucose levels, the cholesterol levels, and the electrolytes (Na and K) levels are all within the normal ranges (Figures 8A–D) and Supplementary Table S2. Next, we performed pharmacokinetics analysis of the MYC-mRNA drug in the serum of the tumor-bearing mice treated with the drug. We found that the drug has a stable half-life in the serum of greater than 24 h (Supplementary Figure S4A).

These data demonstrate that the drug is safe, stable, and well tolerated with no blood dyscrasia, no electrolyte imbalance, no kidney failure, no liver failure, and no pancreatic enzyme function abnormalities.