Telomir-Zn Modulates Intracellular Iron and Copper to Inhibit JmjC Histone Demethylases and Suppress Tumor Growth in Prostate and Triple-Negative Breast Cancer

Materials and Methods Definition and Use of Telomir-Zn

Telomir-Zn (2,4,6-tris(3,4-dihydro-2H-pyrrol-2-yl)pyridine, ZnCl₂) refers to the active pharmaceutical ingredient (API), which was used for all assays, chelation studies, and mechanistic evaluations.

Chelation Assay In Vitro

Ferene based measurements of free Fe²⁺ ions in solutions.

Fe²⁺, Cu²⁺, Zn²⁺ working solutions at 0.8 mM were freshly prepared from FeSO₄, CuCl₂ and ZnCl₂ (Merck # 12354, #C3279, #793523) in HPLC-grade water (J.T. BAKER #4218-03) and filtered through 0.22 µM PES filters. Telomir-Zn (TEL001UK-3) stock at 20 mM, and DFO (Merck #D9533) stock at 15 mM in HPLC-grade water. Prior to use, all chelators were diluted in ammonium acetate (Merck #A1542) buffer (2.5 M, pH 7.2) to a working concentration of 100 µM, maintaining 1% (v/v) ethanol in all conditions to control for solvent effects. Ferene (Merck #82940) working solution was prepared in assay buffer (pH 7.2) to a final Ferene concentration of 2.8 mM.

An Fe²⁺ standard curve was generated in parallel by serial dilution 1:2 (0-800 µM) and used to convert absorbance values to free Fe²⁺ concentrations. Samples containing Fe²⁺ at a constant initial concentration were incubated with increasing concentrations of Telomir-Zn or DFO for 10 min at room temperature. Ferene solution was then added, followed by an additional 10 min incubation to allow chromophore development. Absorbance was measured at 595 nm using a plate reader, and free Fe²⁺ levels were calculated relative to the standard curve.

For the competition assay stock solution was prepared in assay buffer (pH 7.2) to a final Ferene concentration of 2.8 mM. Stock solutions of Telomir-Zn (20 mM in water), and deferoxamine (DFO; 15 mM in water) were diluted in assay buffer containing 1% EtOH to prepare 400 µM working stocks.

Fifty microliters of each test compound (400 µM) were dispensed into wells of a 96-well polypropylene (PP) plate. ZnCl₂ or CuCl₂ solutions (50 µL; 12.5–800 µM) were added to the respective wells and incubated for 10 minutes at room temperature to allow metal– compound interaction. Subsequently, 100 µL of FeSO₄ solution (200 µM) was added to each well. In parallel, FeSO₄ standards (6.25–400 µM) were prepared in the same PP plate.

For quantification of free Fe²⁺, 100 µL Ferene working solution was added to clear, flat-bottom 96-well plates, followed by transfer of 50 µL from each PP-plate reaction well. After incubation for 10 min at room temperature, absorbance was measured at 595 nm using a microplate reader. Free Fe²⁺ concentrations were calculated from the FeSO₄ standard curve.

The Ferene assay generated a concentration-dependent absorbance signal with excellent linearity across the tested range (R² > 0.998). The lowest concentration evaluated (6 µM) was detectable but produced only a weak signal, whereas absorbance values above 0.1 O.D. were consistently observed at 25 µM and higher. For Fe²⁺ standards prepared from FeSO₄, the presence of ascorbic acid did not noticeably affect either signal intensity or linearity.

Fluorescent Measurements

Cell culture and seeding. HaCaT (AddexBio T0020001) keratinocytes were maintained in DMEM (Biowest #L0501) supplemented with 10% fetal bovine serum (FBS) (GIBCO #A5256701), 2 mM sodium pyruvate (Biowest #LO642-100), and 1% penicillin/streptomycin (GIBCO #15140-122). On day 1, cells were seeded into six 96-well plates at a density ensuring subconfluent growth at the time of treatment.

Chelator treatment. On day 2, cells were treated with TelomirZn, or DFO at final concentrations ranging from 0.25 to 50 µM. Vehicle controls were included in parallel. Cells were incubated with compounds for 3, 6, or 16 h at 37°C in a humidified incubator with 5% CO₂.

FerroOrange staining. At each time point, treatment medium was removed and cells were washed three times with Hank’s Balanced Salt Solution (HBSS). FerroOrange (Dojindo Laboratories # F374) at final concentration 1 µM prepared in HBSS was added to each well, and plates were incubated for 30 min at 37°C to allow probe loading and Fe²⁺-dependent fluorescence development.

Fluorescence quantification and imaging. Following staining, fluorescence was first quantified in the 96-well format using a CLARIOstar plate reader (BMG Labtech). Wells were subsequently imaged using a fluorescence microscope at 5×, 10× and 20× magnification. Imaging was performed with excitation at 546 nm and emission collected using a band-pass 575–640 nm filter set. Analysis of IC₅₀ was performed using the software GraphPad Prism.

Cell Culture, Treatment, and Viability Assays

Human triple-negative breast cancer (TNBC) cell lines MDAMB-468, HCC70, HCC1806, and BT-549 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and cultured under the supplier’s recommended conditions.

PC-3 (ATCC CRL-1435) prostate cancer cells were cultured in F-12K; LNCaP clone FGC (ATCC CRL-1740) prostate cancer cells in RPMI-1640 with 2 mM L-glutamine; HL-60 (ATCC CCL240) leukemia cells in RPMI with 15% FBS; HaCaT keratinocytes in DMEM with 2 mM sodium pyruvate. Unless otherwise noted, media were supplemented with 10% FBS and 1% penicillin/ streptomycin. All cell lines were maintained at 37°C in 5% CO₂ and passaged routinely to preserve logarithmic growth. On day 1, cells were seeded into 96-well plates at a density chosen to ensure 20-40% confluence at the time of treatment. On day 3, cells were treated with Telomir-Zn at a range of concentrations prepared in the corresponding culture medium. HL-60 cells in suspension were treated on the day of seeding. Vehicle-treated controls were included in parallel. Cells were incubated with compounds for 48 h prior to endpoint analyses. At the end of treatment, cells were visually inspected and imaged by light microscopy to assess morphology, confluency, and gross cytotoxic effects.

At the end of each incubation period, representative images of all treatments were captured. After imaging, the medium was discarded and replaced with 100 µL of fresh medium.

Cell numbers were calculated from a standard curve generated using untreated cells seeded at known densities under identical assay conditions. Concentration–response relationships were fitted by nonlinear regression in GraphPad Prism to obtain IC₅₀ values. Experiments included technical replicates, and viability was normalized to vehicle controls. Because viability did not reach zero at any tested dose, curve fitting used a fixed theoretical upper concentration of 1000 µM.

Histone Demethylase (KDMs) Assay

Evaluation of the effects of Telomir-Zn on KDMs activities were conducted as previously described (26-27). These assays consist on studying in Sf9 human recombinant cells, expressing the respective enzyme, using as substrates Biotin-H3K4me2, BiotinH3K27me2 or Biotin-H3K36me1 for KDM5s, KDM6s and

KDM2s, respectively.

In Vivo Studies

The mouse is a commonly used species for cancer studies for evaluating the efficacy of new anti-cancerous drugs in accordance with international recommendations and the published literature. The Athymic Nude strain is a well-known laboratory model with sufficient historical data of developing PC-3–derived prostate tumors.

Animals handling was performed according to guidelines of the National Institute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). For this study, mice were housed in individual cages separately (up to 5 per cage) measuring 36.5 x 20.7 x 13 cm with stainless steel top grill facilitating pelleted food and drinking water in plastic bottle; bedding: steam sterilized clean paddy husk (Envigo, Teklad, Laboratory grade, Sani-chips). Bedding material was changed along with the cage at least twice a week. This study was performed in compliance with “The Israel Animal Welfare Act” and following “The Israel Board for Animal Experiments” Ethics Committee approval # NPC-Ph - IL - 2412 – 524.

PC-3 cell injection was performed under inhalational isoflurane anesthesia (3%). A total of 84 mice were utilized and divided into six groups of fourteen animals in each group (10+4 satellite). The number of groups and the total number of animals was based on previous studies and specific guidelines demonstrating that this was the minimum number of animals sufficient to obtain indicative/significant information. Animals were randomized to treatment groups based on tumor volume prior to dosing. Tumor measurements were performed in a blinded manner.

Animals were fed ad libitum a commercial rodent diet (Teklad Certified Global 18% Protein Diet, Envigo cat# 2018SC). Animals had free access to sterilized and acidified drinking water (pH between 2.5 and 3.5) obtained from the municipality supply and treated according to Pharmaseed’s SOP No. 214: ‘Water system”.

Animals were housed under standard laboratory conditions, air conditioned and filtered (HEPA F6/6) with adequate fresh air supply (Minimum 15 air changes/hour). Animals were kept in a climate-controlled environment. Temperatures range was 19–26°C and relative humidity range 35–70% with a 12-hours light and 12-hours dark cycle (6AM/6PM).

Allocation to treatment groups was carried out 10 days post cells injection based on tumors growth to achieve as much as possible balanced groups. The study was conducted in one cycle. Mice were injected with 1x10⁶ PC-3 cells subcutaneously into the right flank region of each mouse, and when the tumors volume reached around 100 mm³, the mice were allocated to groups and began receiving treatment. Six mice (satellites) were sacrificed at this point. Following 12 days of treatment, additional three mice from each group (the satellites) were sacrificed and tumors were collected. Study ended on Day 21 post treatment and tumors were collected from all mice. Animal received vehicle (40% HPβCD+5% DMSO in water), 10 or 100 mg/kg orally at 7.5 mL/kg; PTX (without or with 10 mg/kg of Telomir-Zn) at 20 mg/kg at 10 or 7.5 mL/kg Ip or Po, respectively; or rapamycin at 5 mg/kg at 7.5 mL/kg PO.

Tumors were measured every other day for length (L) and width (W) using a digital caliper starting from tumor appearance. Tumor volume (in mm3) was calculated according to the equation (Length × Width² ×0.5).

At study termination, animals were euthanized using CO₂ inhalation in accordance with approved protocols.

Zebrafish Xenografts

Transgenic Tg(fli1:EGFP)y1 zebrafish embryos were raised at 28 °C for 48 h in E3 embryo medium supplemented with 0.2 mM 1-phenyl-2-thiourea (PTU). Unfertilized eggs or larvae that appeared unhealthy or exhibited developmental defects were excluded prior to the start of the experiment.

Cancer cells were labeled with the red fluorescent dye DiI. Approximately 500–700 labeled cancer cells were subcutaneously implanted into the perivitelline space of 2-day-old embryos. Embryos in which tumor cells were inadvertently injected into the circulation or incorrectly implanted in the yolk rather than the perivitelline space were excluded from the study.

Selected tumor-bearing embryos were sorted into experimental groups (50 embryos per group), and images of primary tumors were acquired immediately after tumor implantation. Tumor-bearing embryos were incubated in E3/PTU medium in the presence of paclitaxel and Telomir-Zn for 3 days at 35.5 °C, with treatment renewed daily.

After the incubation period, images of primary tumors and tails were captured using a red fluorescent filter. Embryos that died or were otherwise lost during the study were excluded from the final analysis.

Images obtained immediately after implantation (day 0) and after 72 h of incubation (day 3) were analyzed using in-house developed software. Primary tumor regression was calculated and normalized to the negative treatment group. The number of disseminated tumor cells in the caudal venous plexus (CVP) after 3 days was manually counted.

Telomere Length Analysis

Absolute human telomere length analysis from tumor tissue of sacrificed satellite mice at 10- and 21-days timeline was determined by using: Absolute Human Telomere Length Quantification qPCR Assay Kit according to the manufacturers kit protocol. ScienCell's Absolute Human Telomere Length Quantification qPCR Assay Kit is designed to directly measure the average telomere length of a human cell population. The telomere primer set recognizes and amplifies telomere sequences. The single copy reference (SCR) primer set recognizes and amplifies a 100 bp-long region on human chromosome 10 and serves as a reference for data normalization. The reference genomic DNA sample with known telomere length serves as a reference for calculating the telomere length of target samples. The carefully designed primers ensure: (i) high efficiency for trustworthy quantification; and (ii) no nonspecific amplification. Each primer set has been validated by qPCR with melt curve analysis for amplification specificity and by template serial dilution for amplification efficiency.

DNA Methylation Analysis

Human quantification of DNA methylation analysis from tumor tissue of sacrificed satellite mice at 10 and 21 days was determined using: OneStep PLUS qMethyl™ Kit according to the manufacturers kit protocol. The OneStep PLUS qMethyl™ PCR Kit was intended for the quantification of human DNA methylation at custom-selected genomic regions. The same DNA sample was analyzed in parallel with the Test PreMix and the Reference PreMix. The Test PreMix contains enzymes that selectively digest unmethylated DNA while leaving methylated DNA intact. Only methylated DNA were amplified in the “Test Reaction.” The Reference PreMix does not contain those enzymes, therefore both methylated and unmethylated DNA are amplified in the “Reference Reaction.” The difference in Cycle threshold (Ct) values between the Reference and Test reactions was used to calculate the percentage of DNA methylation at the selected genomic region. Data represent mean ± SEM of 4–9 tumor DNA samples per group. While initially 10 mice were studied per group, not all tumor samples were sufficiently large to extract the DNA and the number of samples are therefore variable.

Statistical Analysis

Data are presented as mean ± standard error of the mean (SEM) unless otherwise indicated. For Mice studies, group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. For tumor volume, telomere length, and DNA methylation analyses, treatment groups (vehicle, Telomir-Zn 10 mg/kg, Telomir-Zn 100 mg/kg, paclitaxel, TelomirZn + paclitaxel, and rapamycin) were compared at the indicated time points. Statistical analyses were performed using GraphPad Prism (version 10.5.0; GraphPad Software, San Diego, CA).

For Zebrafish studies, normality testing was performed prior to statistical analysis, followed by appropriate parametric or nonparametric tests, including one-way ANOVA or Kruskal–Wallis tests, followed by two-tailed Student’s t-test or Mann–Whitney tests where specified.

Discussion

Telomir-Zn represents a mechanistically differentiated approach that exploits convergent vulnerabilities in iron metabolism and epigenetic regulation. By rapidly depleting labile Fe²⁺ and inhibiting multiple JmjC histone demethylases, it promotes accumulation of activating histone marks, antagonizes DNA methylation at tumor-suppressor promoters, and induces growth arrest or death preferentially in iron-addicted cancer cells.

The iron-rescue data in TNBC lines and the observed promoter demethylation in prostate tumors provide orthogonal evidence linking metal modulation to epigenetic reprogramming. Heterogeneous responses across TNBC models and the stronger anti-metastatic effect in HCC1806 highlight the importance of tumor-specific metabolic and epigenetic context, supporting biomarker-driven patient stratification based on labile iron pools, KDM expression, or iron-handling gene signatures.

Iron chelation is a known therapeutic strategy, with deferoxamine (DFO) as a benchmark due to its high iron affinity [25, 28, 29]. This study shows that Telomir-Zn acts as metal-dependent epigenetic modulator distinct from DFO. While DFO has high Fe²⁺ affinity, Telomir compounds show moderate Fe²⁺ affinity, preferential Cu²⁺ binding, and minimal Zn²⁺ interaction. This suggests they function as redox-metal modulators rather than strict chelators.

Despite lower Fe²⁺ affinity, Telomir-Zn depletes intracellular Fe²⁺ more rapidly, highlighting the importance of cellular permeability. Unlike DFO, Telomir compounds engage intracellular Fe²⁺ quickly, aligning with their early FerroOrange kinetics. Effective intracellular modulation thus depends on accessibility, not just binding strength.

Their rapid Fe²⁺ depletion led to selective cytotoxicity in irondependent malignancies, with iron-rescue experiments confirming Fe²⁺ depletion as the antiproliferative driver. In this context, irondependent tumors are those with elevated labile iron and reliance on Fe²⁺-dependent enzymes. In vivo, oral Telomir-Zn suppressed PC-3 tumor growth with favorable tolerability, consistent with a progressive epigenetic reprogramming mechanism. Importantly, telomere length was unaffected, suggesting no telomerase or replicative stress involvement.

Copper’s role in oncogenesis [8, 30] further supports Telomir-Zn’s dual Fe/Cu targeting, which may be advantageous in cancers where both metals play a role. A key finding is Telomir-Zn’s reduction of promoter hypermethylation in tumor-suppressor genes, aligning with inhibition of Fe²⁺-dependent Jumonji C demethylases. By limiting Fe²⁺ availability, Telomir-Zn reprograms epigenetics without broad chromatin disruption, unlike DNMT or HDAC inhibitors.

Intracellular iron concentrations are substantially higher than those of copper, with iron present in micromolar pools (including both labile and protein-bound fractions), whereas copper is maintained at much lower, tightly regulated levels due to its high redox reactivity [31, 32]. These quantitative differences are critical for interpreting the dual metal-modulating properties of Telomir [33, 34]. In this context, we believe that the biological impact of Telomir is not solely determined by total metal abundance but rather by its interaction with the labile, bioactive metal pools that regulate enzymatic activity and redox signaling. While iron modulation is likely the dominant driver of effects on Fe²⁺-dependent enzymes such as JmjC histone demethylases, even small perturbations in labile copper can have disproportionate effects on redox balance, mitochondrial function, and copper-dependent enzymes due to the tight homeostatic control of this metal. Thus, the apparent dual activity of Telomir may reflect differential but complementary modulation of iron- and copper-dependent pathways, rather than equivalent quantitative targeting of both metals.

The present study demonstrates that Telomir-Zn exerts antitumor activity across multiple TNBC and other cancer models, with heterogeneous responses observed between cell lines. The most direct mechanistic observation in this dataset is the rescue of Telomir-Zn cytotoxicity following Fe²⁺ supplementation, suggesting an iron-dependent component of the compound’s activity.

The observed reduction in promoter DNA methylation following Telomir treatment is mechanistically consistent with inhibition of Fe²⁺-dependent Jumonji C histone demethylases (KDMs). These enzymes regulate key histone methylation marks that control chromatin accessibility and transcriptional activity [35, 36]. Depletion of intracellular labile Fe²⁺ impairs KDM catalytic function, leading to accumulation of activating histone marks such as H3K4me3 and H3K36me2. These chromatin changes are known to antagonize DNA methylation by limiting recruitment and activity of DNA methyltransferases (DNMTs), which preferentially target transcriptionally repressed and H3K4-unmethylated regions. In parallel, the establishment of a more permissive chromatin environment promotes transcriptional activation and facilitates passive and active DNA demethylation processes, including reduced maintenance methylation during replication and enhanced accessibility to TET-mediated oxidation pathways [37, 38]. Consistent with this framework, Telomir treatment resulted in decreased promoter methylation across multiple tumor suppressor genes, including STAT1, GSTP1, RASSF1A, CDKN2A, and MASPIN. These findings support a model in which modulation of intracellular metal availability induces coordinated epigenetic reprogramming, linking metabolic control of Fe²⁺-dependent enzymes to both histone and DNA methylation landscapes. Unlike direct DNMT inhibitors, this mechanism enables simultaneous regulation of multiple chromatin-modifying pathways, providing a broader and potentially more physiologic strategy to reverse epigenetic silencing in iron-dependent malignancies.

However, given the central roles of iron and copper in redox biology and cellular metabolism, these effects are unlikely to be exclusively epigenetic. Modulation of labile Fe²⁺ and Cu²⁺ pools can alter reactive oxygen species dynamics, mitochondrial function, and metal-dependent enzymatic processes, all of which may contribute to the observed anti-tumor activity. The partial rescue of cytotoxicity by exogenous iron further supports a broader dependence on metal availability that integrates metabolic and epigenetic vulnerabilities. Together, these findings support a model in which Telomir induces coordinated epigenetic reprogramming in parallel with redox and metabolic modulation, with these processes acting in an interconnected manner rather than as independent mechanisms.

The zebrafish xenograft experiments further support the translational potential of Telomir-Zn by demonstrating antitumor activity in vivo. Zebrafish xenograft models have emerged as powerful preclinical tools capable of rapidly evaluating therapeutic response and metastatic dissemination [39, 40].

The enhanced activity observed in combination with paclitaxel suggests that modulation of metal homeostasis may sensitize tumors to cytotoxic chemotherapy. Future studies should investigate whether Telomir-Zn influences oxidative stress pathways, ferroptosis susceptibility, or iron-dependent chromatin regulation [41-43].

This study further indicates that Telomir-Zn elicits a heterogeneous but biologically coherent antitumor response across breast cancer xenograft models, driven by cell line–dependent molecular vulnerabilities rather than uniform cytotoxicity. Sensitivity to Telomir-Zn in HCC1806 and BT-549 xenografts may be associated with dysregulated iron handling, characterized by elevated labile Fe²⁺ pools, limited ferritin buffering capacity, and increased basal oxidative stress, which together may confer susceptibility to redox imbalance induced by iron and copper chelation.

In these models, disruption of Fe²⁺-dependent JmjC histone demethylases may further compromise epigenetic plasticity, leading to transcriptional instability, replication stress, and growth arrest or cell death, with a potential contribution of ferroptotic mechanisms in BT-549. In contrast, MDA-MB-231 xenografts may exhibit resistance due to enhanced antioxidant buffering, adaptive metabolic programs, and compensatory signaling networks, including KRAS–STAT3 pathways, which together may limit sensitivity to metal-dependent therapeutic stress [7, 44, 45].

The current in vivo study in mice was conducted in athymic nude mice, which lack functional T cells and therefore do not fully capture the eventual contribution of adaptive immunity to tumor progression and therapeutic response. This limitation is particularly relevant in the context of metal biology, as iron and copper homeostasis are closely linked to immune cell function, inflammatory signaling, and tumor–immune interactions. Modulation of labile Fe²⁺ and Cu²⁺ may therefore influence not only tumor cell–intrinsic pathways but also components of the tumor microenvironment, including innate and adaptive immune responses, which are not represented in this model [46-48]. The data of TNBC cells in zebrafish, an established in vivo system that enables interrogation of whole-organism redox biology, metal homeostasis, and immune–metabolic interactions. TelomirZn significantly reduced primary tumor size in BT-549 and HCC1806 xenografts and decreased metastatic dissemination in HCC1806. These findings support a broader, systemic effect of metal modulation consistent with the mechanisms described here, although the translational relevance to mammalian tumor– immune interactions remain to be fully defined. Accordingly, the anti-tumor effects observed in the current mice study should be interpreted primarily as tumor cell–autonomous, and further evaluation in immunocompetent and syngeneic mammalian models will be required to delineate the contribution of immunemediated mechanisms and their potential therapeutic implications.

Emerging evidence supports the concept of iron addiction, whereby subsets of cancers, including TNBC, reprogram iron metabolism to increase iron uptake, retain intracellular iron, and expand the labile iron pool in order to sustain proliferation, redox balance, and epigenetic plasticity. In the literature, this phenotype is generally inferred from a combination of features rather than a single marker, including increased CD71 expression, reduced ferroportin-mediated iron export, higher intracellular labile iron, and preferential sensitivity to iron depletion or chelation [4951]. In this context, inhibition of Fe²⁺-dependent JmjC histone demethylases represents a plausible downstream vulnerability, disrupting chromatin dynamics and contributing to transcriptional instability, replication stress, and growth arrest or cell death. These effects may be amplified in more iron-dependent tumors and, in some settings, may intersect with ferroptotic vulnerability. In contrast, less sensitive models such as prostate or pancreatic cells may show relative resistance through lower reliance on labile iron, stronger antioxidant buffering, greater metabolic adaptability, or compensatory signaling pathways.

The preferential affinity of Telomir-Zn for Cu²⁺ over Fe²⁺ is particularly relevant in TNBC, where copper plays a critical role in metastatic progression. Beyond its well-established functions in mitochondrial energy metabolism, copper drives multiple steps of the metastatic cascade, including extracellular matrix remodeling via LOX, modulation of the pre-metastatic niche, and maintenance of cancer stem cell properties [22, 23]. The anti-metastatic effects observed with Telomir-Zn in the HCC1806 zebrafish model align with prior findings that copper depletion selectively impairs metastatic dissemination while sparing primary tumor proliferation.

This dual modulation of labile iron and copper pools by TelomirZn may therefore offer complementary advantages over iron-only chelators, simultaneously targeting epigenetic regulation (via Fe²⁺dependent KDMs) and mitochondrial/metastatic pathways (via Cu²⁺).

Collectively, these findings suggest that Telomir-Zn may preferentially target tumors with features consistent with iron dependence and epigenetic liability, supporting stratification using established iron-metabolism markers rather than drug response alone.

Conclusion

Selective intracellular Fe²⁺/Cu²⁺ modulation by Telomir-Zn drives epigenetic reprogramming and exerts antitumor activity in preclinical models of prostate cancer and TNBC. This approach offers a novel therapeutic strategy for targeting metabolic-epigenetic vulnerabilities in aggressive, iron-addicted malignancies.

Abbreviations

API: Active pharmaceutical ingredient

BW: Body weight

CARD: caspase activation and recruitment domain

DFO: Deferoxamine

DNA: Deoxyribonucleic acid

DNMT: DNA methyltransferase

EMT: Epithelial–mesenchymal transition

FBS: Fetal bovine serum

FPN: Ferroportin

HDM: Histone demethylase

IC₅₀: Half-maximal inhibitory concentration

JmjC: Jumonji C domain

KDM: Lysine demethylase

LIP: Labile iron pool

PC-3: Human prostate cancer cell line

PTX : Paclitaxel

PYCARD/ASC:   Apoptosis-associated Speck-like protein containing a CARD qPCR: Quantitative polymerase chain reaction

ROS: Reactive oxygen species

SEM: Standard error of the mean

STAT1: Signal transducer and activator of transcription 1

TNBC: Triple-negative breast cancer

UTX (KDM6A): Ubiquitously Transcribed Tetratricopeptide Repeat on X Chromosome

XTT/WST-1:Tetrazolium-based cell viability assays.

Data Availability: All data generated and analyzed in this study are included in the manuscript and its accompanying figures. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics Statement: All animal studies were conducted in accordance with institutional guidelines for the care and use of laboratory animals. The PC-3 xenograft study was performed under an approved Institutional Animal Care and Use Committee (IACUC) protocol at Pharmaseed (Nes-Ziona, Israel). All procedures—including tumor implantation, dosing, monitoring, and euthanasia—were performed in accordance with internationally accepted ethical standards for animal research. This study was performed in compliance with “The Israel Animal Welfare Act” and following “The Israel Board for Animal Experiments” Ethics Committee approval # NPC-Ph - IL - 2412 – 524.

Author Contributions: I.A. and E.A. conceived and designed the study. M.G. and R.M. performed the in vitro assays and chelation studies. P.R.H. conducted the in vitro assays and in vivo mouse xenograft experiments. G.V.R and A.E. performed the Zebrafish in vivo experiments. I.A. wrote the manuscript with input from all authors. All authors reviewed and approved the final version of the manuscript.

Funding: This research was supported by Telomir Pharmaceuticals, Inc., which provided funding for in vitro studies, analytical development, and in vivo xenograft experiments.

Acknowledgments: The authors thank the scientific and technical teams at Pharmaseed, Smart Assays Biotechnologies and BioReperia AB for their contributions to the in vivo and in vitro studies. We also acknowledge the support of Telomir Pharmaceuticals, Inc. for project coordination and study oversight.

Conflict of Interest: Erez Aminov is the Chief Executive Officer of Telomir Pharmaceuticals, Inc. Itzchak Angel and other co-authors are consultants, or scientific advisors to the company. Telomir Pharmaceuticals is developing Telomir-Zn. All other authors declare no additional competing financial interests. E.A. is the founder and Chief Executive Officer of Telomir Pharmaceuticals, Inc., I.A. is Chief Scientific Advisor to Telomir Pharmaceuticals and. M.G. and R.M. are employees of Smart Assays Biotechnologies; P.R.H. is an employee of Pharmaseed Ltd.; and G.V.R. and A.E. are employees of BioReperia AB — contract research organizations that received funding from Telomir Pharmaceuticals to conduct the studies described. Telomir Pharmaceuticals is the sponsor of this research and is developing Telomir-Zn, which is covered by the company’s intellectual property. The sponsor participated in the study design, data interpretation, and preparation of this manuscript. Beyond the disclosures above, the authors declare no further competing interests.

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