Functional Gene Restoration and Cancer Stem Cell Eradication in a Treatment-Resistant TNBC Case: A First-in-Human Paradigm Shift

Authors

Corresponding Author: Marc Malone,

Dr. Erin Greer, Dr. Trisha Wimbs (provided clinical peer-review)

Disclaimer: This is not a treatment protocol. This is not a supplement stack. This is a new biological reprogramming system. This breakthrough was a one-of-a-kind emergency intervention in a last-resort context with experimental variables. Do not attempt to replicate this without deep scientific understanding and supervision. This is for further research only (as is written in the paper), it is not generalizable or for treatment until clinically rolled out under controlled conditions.

ABSTRACT

Triple-negative breast cancer (TNBC) is among the most lethal, treatment-resistant cancer types, lacking hormone receptors and driven by aggressive cancer stem cell (CSC) signaling. We report the first documented human case of multi-locus gene reprogramming, restoration and CSC molecular pathway eradication; achieved without gene editing, viral vectors, or cytotoxic agents. This dual intervention, delivered under a compassionate-use model, resulted in reactivation of silenced estrogen and progesterone receptor genes; ESR1 (6q25.1-q25.2) and PGR (11q22-q23) from undetectable to 25% and <1% to 15%, respectively, within three weeks with Tumor volume declining -70% and complete disappearance of one metastatic lymph node, signaling systemic effect. Then CSC pathways NOTCH and GLI2, known causes of recurrence and metastasis across cancers, were fully suppressed to undetectable levels. These molecular shifts occurred in a patient with ultra-lethal metastatic TNBC (TP53, PMS2, KMT2C mutations; Ki-67 >90%) who failed all conventional treatments, including chemotherapy, immunotherapy, and targeted agents (phase II).

The phase I therapeutic system integrated three mechanistic pillars and led to an unprecedented tripled survival rate:

1. A Novel Triune Restoration Framework: PI3K-mTOR decoupling, SIN3-HDAC inhibition, and α-ketoglutarate (a-KG)-induced chromatin remodeling enabling transcriptional derepression.

2. Metabolic Stress Induction: Glycolysis/glutaminolysis inhibition sustained by nutraceutical and fasting-based ATP flux rewiring.

3. Chloride Channel Activation & Signal Disruption: Inducing apoptosis and halting EGFR/PI3K oncogenic signaling (streptomyces-avermectins).

Within two weeks of phase III protocol delivery, mutational burden was reduced: TP53 from 31.6% to 16% (49% drop), KMT2C 3.8% to 1.4% (63% drop), PMS2 from 50.3% to 44.7%, and Ki-67 from 100% to 65%, and circulating tumor cells (CTCs) dropped by 27%. Niclosamide, administered at a late terminal stage, fully suppressed CSC master pathways NOTCH2 and GLI2 axes, supporting the case’s functional eradication, selected despite being off-market due to safety profile and moiety in reprogramming potential. While CSC suppression occurred too late to reverse fatal tumor infiltrated-organ damage, it marks the first human documentation of targeted total CSC pathway inhibition. Full CSC suppression with demonstrated systemic mutational and proliferative collapse, is the closest to a functional cancer cure model (differing from ‘absolute cure’) recorded in a human being due to pan-cancer potential, no toxicity, and causal inhibition of oncogenic signal pathways driving disease.  

Mechanistically, this novel Triune Restoration Framework demonstrates a reproducible route to gene reactivation through:

→PI3K inhibition preserving mTOR-S6 signaling repair 

→SIN3-HDAC complex disruption

→a-KG-mediated histone and DNA demethylation

→HDAC-DNMT-RNR remodeling driving chromatin accessibility

The result is the first documented re-expression of multiple functional genes across separate chromosomes in a living human - an achievement not accomplished by CRISPR or any prior method, marking one of the most significant, initial biological breakthroughs ever recorded. Neurological symptoms (visual distortion, sensory shifts) aligned with reactivation of Retinoic Acid Receptors in SIN3-HDAC TNBC preclinical models, indicating CNS involvement.

Clinically, this translated to a tripled survival rate (average 3.8 months → 12 months), with <1% surviving past the baseline - unprecedented in a profile recorded as one of the most ultra-lethal in oncology & medicine. Molecular outcomes were validated by notable independent laboratories and imaging.

These results redefine the boundaries of epigenetic therapy, CSC-targeted oncology, and regenerative medicine. They suggest that functional reprogramming of silenced genes and eradication of recurrence driving stemness pathways can be achieved in vivo using FDA-approved compounds and metabolic modulation. This Biological Systems Engineering approach warrants further investigation for applications across cancer, neurodevelopmental, degenerative, and endocrine disorders.

INTRODUCTION

Cancer is the second-leading cause of death worldwide, with many rarer and more aggressive subtypes still lacking in Approved and Targeted treatments. Triple-Negative Breast Cancer (TNBC) is considered one of the most aggressive cancers, with few treatment options, a high mortality rate of 84-92% in 5 years, and median overall survival of just 10.2 months in many cohorts [1, 2]. TNBC accounts for only 10-15% of all breast cancer cases and over half the deaths. TNBCs lack estrogen and progesterone receptors and express low or no HER2, and therefore do not respond to hormonal therapies. TNBC is the most aggressive and treatment-resistant form of breast cancer, hence its poor prognosis compared to other breast cancers and a 90% treatment failure rate with chemotherapy regimens [3]. TNBC is chemotherapy sensitive due to a typically high KI-67 expression, its efficacy in generating disease-free survival however has limited benefit due to resistance and high cancer stem cell load [4]. Advances have been made such as those with biomarkers PDL1 for Anti-PD1 immunotherapy. However, only a minority of these patients respond to immune checkpoint or PARP inhibitors and upon response often develop resistance and recurrence.

The repression of genes such as ESR1 (estrogen receptor alpha) and PGR (progesterone receptor) involves a complex interplay between metabolic pathways, particularly (aerobic) glycolysis, and epigenetic modifications like H3K27me3. Additionally, this interplay affects other molecular markers we will include in our sample, including histone deacetylases (HDACs), DNA methyltransferases (DNMTs), ribonucleotide reductases (RNRs), and the mTOR-S6 signaling pathway. All of these are involved in repression or expression of epigenetic modification.

Glycolysis and H3K27me3 in Gene Repression

Glycolysis influences the cellular levels of metabolites that are crucial for both proliferation and epigenetic modifications. For instance, the availability of Acetyl-CoA, a product of glucose metabolism, is essential for histone acetylation. Reduced Citrate and Acetyl-CoA’s shift in metabolism reduces citrate biosynthesis, and because citrate is a precursor to acetyl-CoA, its reduced production favors a deacetylation state of proteins, which can contribute to tumor aggressiveness [5]. A reduction in acetyl-CoA levels can lead to decreased histone acetylation and increased deposition of repressive marks like H3K27me3, thereby silencing gene expression. Studies have shown that metabolic stresses, such as serine starvation, can reprogram glucose metabolism, leading to reduced acetyl-CoA generation and subsequent repression of ERα expression through increased H3K27me3 levels at the ESR1 promoter [6].

Impact on ESR1 and PGR Repression

The increase in H3K27me3 at the promoters of ESR1 and PGR genes results in a condensed chromatin structure, hindering the access of transcriptional machinery and leading to gene silencing [7]. This epigenetic repression diminishes the expression of estrogen and progesterone receptors, which are critical for normal cellular responses to hormonal signals.

Effects on Molecular Markers, Sampled as Active in Reprogramming

Histone-Deacetylase (HDACs) Dipeptide: The SIN3-HDAC complex is involved in deacetylating histones, contributing to chromatin condensation and gene repression. The presence of H3K27me3 can recruit HDAC-containing complexes, reinforcing the repressive chromatin state. HDAC-Dipeptide inhibits this condensation allowing for genetic expression to be active from inactive.

DNA-Methylstransferases (DNMTs): DNA methyltransferases add methyl groups to DNA, leading to gene silencing. The interaction between DNA methylation and H3K27me3 can synergistically repress gene expression, as seen in the repression of certain genes in cancer cells.

Ribonucleotide Reductase (RNRs): Ribonucleotide reductases are primarily involved in DNA synthesis and repair, their expression can be influenced by the epigenetic landscape, affecting cell proliferation and response to metabolic changes.

mTOR-S6 Pathway: The mechanistic target of rapamycin (mTOR) pathway is sensitive to cellular energy status and nutrient availability. Modulations in mTOR activity can influence many epigenetic processes including histone modifications such as H3K27me3, thereby impacting gene expression patterns related to cell growth and metabolism.

The metabolic state of a cell, particularly through glycolysis, significantly impacts the epigenetic regulation of genes like ESR1 and PGR. Alterations in metabolite availability can lead to increased H3K27me3 deposition, recruiting repressive complexes such as SIN3-HDAC and DNMTs, resulting in chromatin condensation and gene silencing. This intricate network underscores the importance of metabolic-epigenetic crosstalk in regulating gene expression and cellular function.

Current Limitations with Gene-Therapy

CRISPR offers an extremely sophisticated mechanism of action and has provided groundbreaking insights and results. This strategy addresses some possible challenges often encountered in gene editing by ensuring that the cellular milieu supports and maintains the desired genetic modifications to not produce adverse “off-target” events, so it is not rejected by endogenous gene programming and the immune system, i.e., a foreign entity entering the genome. The interplay between metabolic, biochemical processes and epigenetic modifications is crucial for effective gene expression changes in humans. Metabolic factors, including nutrient availability, can influence the activity of enzymes responsible for DNA methylation and histone acetylation, thereby creating an environment conducive to stable epigenetic reprogramming. Even CRISPR, despite its precision, often fails to induce stable chromatin-level activation in somatic tissue without extensive remodeling of the surrounding transcriptional landscape. This is especially vital when looking at multi-chromosome gene recovery, not yet documented in editing or any other human case.

Our results, demonstrating modulation of molecular markers such as DNA methyltransferases (DNMTs), ribonucleotide reductase (RNR), histone deacetylases (HDACs), and the mammalian target of rapamycin (mTOR) pathway, provide empirical support for this approach. The observed reactivation of genes: estrogen receptor (ER),

progesterone receptor (PR), and clinically-symptomatic with previous lab evidence expression of retinoic acid receptor beta (RARB) in the same PAH2-SIN3-HDAC mechanism of action [8], further underscores the effectiveness of integrating metabolic interventions with epigenetic strategies. DNMTs in the SIN3-HDAC complex have been cited as highly relevant markers in genetic reactivation/re-expression of ER and RARB in cancer models while also their drug targets have been lacking effectiveness [9].

This comprehensive method not only facilitates gene reprogramming but also offers a promising avenue for therapeutic interventions in endocrine and degenerative disorders. By creating a supportive metabolic environment, this approach enhances the stability and efficacy of epigenetic modifications, potentially leading to more reliable and enduring therapeutic outcomes while most critically, avoiding an aberrant genetic or immune response.

Glycolysis and histone modifications such as H3K27me3 influence the repression of molecular markers, and the reactivation of inactive genes is pivotal to understanding the metabolic-epigenetic interplay in gene regulation.

Glycolysis-Glutaminolysis Programming Histone Repression

Glycolysis, the metabolic pathway that converts glucose into pyruvate, not only provides energy but also generates metabolites that serve as substrates or cofactors for epigenetic enzymes. The histone mark H3K27me3, a trimethylation at the 27th lysine residue of histone H3, is associated with transcriptional repression and is deposited by Polycomb Repressive Complex 2 (PRC2). H3K27me3 has a clear integration with metabolic reprogramming via Glycolysis-Glutaminolysis [10]

Studies have shown that aging leads to a drift in H3K27me3 distribution, resulting in reduced expression of glycolytic genes. This reduction adversely affects energy production and cellular redox states, highlighting a link between H3K27me3 and glycolytic activity. Conversely, a decrease in H3K27me3 levels, due to PRC2 deficiency, has been associated with enhanced glycolysis and improved lifespan in model organisms [11]. In aerobic glycolysis cells (cancerous) we see the same level of interconnected metabolic-histone networks, where pyruvate produced is converted to lactate and results in ‘histone lactylation’, the repression or activation of histone marks.

Reversal of Gene Inactivation through Metabolic and Epigenetic Modulation

The reactivation of previously silenced genes involves both the removal of repressive epigenetic marks and the establishment of an active chromatin state. Metabolic shifts can influence this process:

α-Ketoglutarate (α-KG): This tricarboxylic acid (TCA) cycle intermediate acts as a cofactor for Jumonji domain-containing histone demethylases, which can demethylate H3K27me3, leading to gene activation.

Histone Acetylation: Acetyl-CoA, another metabolite, serves as a substrate for histone acetyltransferases. Acetylation of histones neutralizes their positive charge, reducing their affinity for DNA and resulting in a more open chromatin structure conducive to transcription.

Therefore, metabolic interventions that increase α-KG and acetyl-CoA levels, while inhibiting Glycolysis-Glutaminolysis in cancer specifically, can promote the removal of repressive marks like H3K27me3 and enhance histone acetylation, facilitating the reactivation of silenced genes [12].

The interplay between glycolysis and histones play a significant role in the regulation of gene expression. Metabolic shifts that affect the availability of key metabolites can modulate epigenetic landscapes, leading to the repression or activation of specific genes. Understanding these connections offers potential avenues for therapeutic interventions aimed at reprogramming gene expression patterns in various diseases safely.

Overview of the metabolic-to-epigenetic pathway discovered

Glycolysis-Glutaminolysis switch to a-KG → SIN3-HDAC inhibition → Histone Repression (e.g H3K27me3) loss → ESR1/PGR gene re-expression (limited/partial)

The metabolic shift from glucose and glutamine to α-ketoglutarate (α-KG) influencing H3K27me3 repression loss, thereby supporting histone acetylation and epigenetic reprogramming, is grounded in the intricate interplay between metabolism and epigenetics [13]. Underexpression of ESR1 and lack of PGR when a-KG promotion (fasting with the nutraceuticals) ended, allows us to reverse engineer its significant - but limited/partial impact while also reinforcing the necessity that a complete metabolic-to-epigenetic system requires dysregulated (oncogenic) signal pathway inhibition to reinforce mTOR’s repair mode, with emphasis on not inhibiting mTOR in the process. This treatment’s additional SIN3-HDAC inhibition supported further acetylation dynamics.

Metabolic Shift to α-Ketoglutarate and Epigenetic Modifications

Controlled-Fasting, accompanied by supplemental Glucose and Glutamate modulators and FDA-approved apoptosis inducing drugs with HDAC complex inhibitory activity, helped facilitate the epigenetic reprogramming of the cells. Controlled-Fasting was chosen as the method to ‘starve Glutamine’ from the cancer cells because there’s no safe and effective drug available for prescription that can pharmacologically block Glutamine. Glucose starvation was a much easier dietary uptake. We tested Metformin, but it had little effect comparatively. Studies suggest Glutamine-starved cells convert to a-KG supporting histone deacytelation and mTOR signaling, which is confirmed in our genetic samples [14].

Exploration into the metabolic shift towards α-ketoglutarate (α-KG) and its impact on the HDAC-SIN3 complex provides valuable insights into the mechanisms of epigenetic reprogramming, and how this metabolic alteration supports the observed molecular demethylation and chromatin modification markers.

α-Ketoglutarate and Histone Demethylation

α-KG serves as a crucial cofactor for Jumonji C (JmjC) domain-containing histone demethylases, such as UTX/KDM6A and JMJD3/KDM6B. These enzymes specifically target the H3K27me3 mark. Elevated levels of α-KG enhance the activity of these demethylases, leading to the removal of methyl groups from H3K27me3. This demethylation results in a more relaxed chromatin structure, facilitating gene activation [15].

Interplay with the HDAC-SIN3 Complex

The SIN3 complex, in association with histone deacetylases (HDACs), plays a pivotal role in chromatin remodeling and gene expression regulation. HDACs remove acetyl groups from histone tails, leading to chromatin condensation and transcriptional repression. However, the demethylation of H3K27me3 mediated by α-KG-dependent demethylases can influence the recruitment and activity of the HDAC-SIN3 complex [16]. Specifically, the reduction of repressive methyl marks can diminish the binding affinity of repressor complexes, thereby reducing their repressive effects and allowing for increased histone acetylation. The metabolic shift in of itself would likely lack the targeting and potency to reverse significant SIN3-HDAC gene repression, but in partnership with effective molecular targeting, in this case via Avermectins, the impact is demonstrable.

Metabolic Histone Derepression, PI3K-mTOR and Gene Reprogramming

The loss of H3K27me3 repression creates a chromatin environment that is more permissive to acetylation, particularly at H3K27ac. This acetylation is associated with active enhancers and is crucial for the transcriptional activation of genes involved in cell identity and function. The dynamic interplay between histone demethylation and acetylation underscores the complexity of epigenetic regulation and highlights the potential of metabolic interventions in modulating gene expression [17].

The metabolic shift towards α-KG not only promotes the demethylation of repressive histone marks like H3K27me3 but also indirectly influences the activity of the HDAC-SIN3 complex. This dual action facilitates a chromatin state that supports histone acetylation and gene activation, thereby contributing to effective epigenetic reprogramming. Understanding these interconnected pathways offers valuable insights into developing therapeutic strategies for diseases associated with epigenetic dysregulation. This could be why such profound epigenetic modifications are not seen in humans that are seen in human cell lines or animal models in labs: sustained functional genetic reprogramming in human beings with entire genome-wide cross communication requires a more systemic approach that recruits support from the extracellular & intracellular environments in coordination with DNA, RNA and Histones.

To extend this foundation into a systems-level intervention, recent literature supports a coherent integration of metabolic and genetic regulatory nodes. Specifically:

1. PI3K as a Negative Regulator of mTOR: While PI3K/AKT signaling is commonly upregulated in oncogenesis, this hyperactivation paradoxically suppresses mTOR-S6 repair signaling due to negative feedback and oxidative stress accumulation. While aberrant mTOR is known to further disease, especially cancer, mTOR-S6 also attenuates negative loops [18].

2. a-KG inhibits PI3K inducing apoptosis: The anti-tumor effect of a-KG promoted cell death in renal cancer cells via mediated ROS-PI3K/Akt/mTOR pathway [19].

3. a-KG supported mTOR-S6 phosphorylation: a-KG supplementation increased mTOR, S6 kinase beta 1 (S6K1) supporting protein-synthesis and tissue repair. [20]

4. a-KG-mediated demethylation SIN3: Epigenetic modification via KDM5A, an α-KG-dependent demethylase, interacts with SIN3-HDAC complex in non-pluripotent cells. TET proteins interact with OGT, which then regulates histone and DNA marks, including H3K4me3, partly by recruiting SIN3 [21].

5. Glycolysis-flux via a-KG and DNMT, HDAC Remodeling: Suppression of glutaminolysis and glycolysis increases intracellular a-KG, supporting a favorable ratio for chromatin derepression, while also upregulating RNR repair and downregulating DNMT and HDAC activity, markers observed in this protocol and consistent with metabolic induction of DNA repair and gene reactivation. Decreased α-KG sees histone hypermethylation, tumor dedifferentiation, and resistance. In vivo studies showed that DNMT1 promotes tumorigenesis in melanoma cells by the direct activation of the PI3K/AKT/mTOR pathway. intra-tumoral increase in α-KG levels attenuated H3K4 tri-methylation downregulated PI3K and MAPK [22].

6. Avermectins Inhibit PI3K: Ivermectin and related avermectins inhibit the PI3K/AKT/mTOR axis, as demonstrated in various cancer and breast cancer models, and are proposed here as facilitating the reprogramming via weighted PI3K but moderate mTOR inhibition, allowing for a-KG facilitated repair, synergistic with SIN3-HDAC inhibition as we’ll discuss (Yeon-Jin Kwon et al.) [23].

7. a-KG and Metabolic PI3K activity: Nutrient restriction led to indirect reduction of oncogenic PI3K pathway activity, and acetylation dynamics. Serine metabolism, Glycolysis-Glutaminolysis inhibition and a-KG modulation ratio could therefore support PI3K ‘decoupling’, and correlated PI3K/AKT involvement in a Glycolysis feedback loop in GBM (and MYC, TP53, EGFR) [24, 25].

Together, these interactions form the mechanistic basis of what became our novel “triune restoration system” described herein, enabling the first human multi-chromosome gene activation ever recorded, done without cytotoxicity or editing, and positioning reprogramming as a viable human therapeutic strategy.

Cancer Stem Cell Genesis and its Developmental Axis

Cancer stem cells (CSCs) represent a subset but highly consequential subpopulation of tumor cells with capabilities for self-renewal, resistance to conventional therapy, and initiation of recurrence while driving metastasis. A substantial body of preclinical research has identified three core developmental signaling pathways: WNT, Notch, and the Hedgehog-GLI2 axis are known molecular originators of CSC programming across a wide range of solid tumors and virtually all known cancers, including TNBC [26, 27]. These pathways transcriptionally regulate CSC surface phenotypes such as CD44, CD133, and ALDH1, which are often used as proxy markers. However, the presence or absence of these

surface antigens is subordinate to the activation state of the upstream signaling axis. WNT-Notch-Hedgehog-GLI2 not only enables CSC survival and immune evasion, but also initiates downstream effects such as hyperproliferation (via KI-67 elevation), CTC dissemination, and upregulation of genomic instability drivers such as TP53 and PMS2, all validated in this case study and significant across cancers [28].

Importantly, this malignant cascade is a bi-directional ‘feedback loop’. As TP53 mutations and proliferative gene signatures accumulate, they further reinforce WNT and Notch signaling, creating a self-sustaining and perpetual CSC presence that drives aggressive tumor progression. Therefore, suppression of this upstream signaling triad is not merely associative but causal to disrupt the CSC lineage and halt downstream tumor aggressiveness and treatment-resistance. In this light, direct molecular silencing of Notch and GLI2 is not just mechanistically meaningful, it precedes surrogate markers such as CD44 or ALDH1 for the purposes of clinical validation. Although extremely rare early-stage malignancies might momentarily evade full WNT, Notch, or Hedgehog-GLI2 engagement, the overwhelming evidence supports these pathways as universal architects of tumor maintenance, CSC programming, and therapeutic resistance across virtually all known human cancers, emphasizing Notch in self-renewal, immune modulation, and therapy escape [29, 30]. Therefore, targeting and eradicating these pathways represents a highly generalizable and profoundly impactful strategy across a vast majority of human cancers; full suppression of these CSC pathways from an oncological perspective would address a fundamental Achilles' heel for most malignancies. This strategy could be the first human-demonstrated ‘pan-cancer’ treatment that goes both to the causal nature of the disease and does so with no patient felt or clinically-observed toxicity.

TNBC’s Aggressive Proliferation and Mutational Burden

The aggressiveness of TNBC is driven by a combination of distinct biological features, including a high CSC load via aberrant activation of the WNT, Hedgehog and particularly Notch signaling pathways, an elevated mutational burden, and dysregulated growth signals, all of which contribute to rapid tumor progression and therapeutic resistance. One of the central features of TNBC is the elevated presence of CSCs, which are observed to drive tumor initiation, metastatic spread, and recurrence. These CSCs exhibit unique properties such as self-renewal, resistance to conventional therapies, and the ability to maintain heterogeneity within the tumor. The interaction between CSCs and critical oncogenic pathways, particularly the WNT and Notch signaling cascade, is pivotal in promoting these malignant traits.

Hyperactivation of PI3K (including PIK3CA mutations) and its downstream effectors significantly enhances cell survival, proliferation, and resistance to apoptotic signals. This pathway via dysregulated mTOR plays a crucial role in the aggressiveness of TNBC, particularly apoptosis-resistance and cancers broadly by promoting both cell proliferation and metastasis [31]. Furthermore, genetic silencing of important tumor suppressor genes such as ESR1, PGR, and RARB in TNBC removes critical regulatory checkpoints that typically control cell cycle progression and proliferation in estrogen receptor-positive (ER+) or HER2+ breast cancers [32, 33]. This silencing leads to unchecked cell proliferation, further exacerbating tumor growth beyond what is seen in typical breast cancers, where these pathways are functional.

The Critical Roles of TP53 and PMS2 in Cancer Progression and Treatment Resistance

TP53 is a paramount tumor suppressor gene, often termed the "guardian of the genome." Its multifaceted roles encompass orchestrating cell cycle arrest, DNA repair, and programmed cell death (apoptosis) in response to cellular stress. When TP53's expression is reduced or its function is abrogated, cells gain the ability to bypass crucial checkpoints, accumulate DNA damage, and evade the apoptotic pathways that normally eliminate aberrant cells. This dysregulation directly drives tumor initiation and progression. Critically, loss of functional TP53 is a well-established driver of aggressive cancers and is a primary mechanism contributing to profound resistance to a wide array of conventional chemotherapies. Many cytotoxic drugs exert their effects by inducing DNA damage, relying on an intact TP53 pathway to trigger apoptosis. Without functional TP53, these therapeutic strategies become significantly less effective, leading to accelerated disease progression and drastically reduced survival rates [34].

PMS2 is a vital component of the DNA mismatch repair (MMR) pathway. This intricate system is indispensable for correcting small errors (mismatches, insertions, or deletions) that inevitably arise during DNA replication. A reduction in PMS2 expression, or any other core MMR component, results in a profound mutator phenotype, where cells accumulate a disproportionately high number of somatic mutations. While a high mutational burden can sometimes correlate with response to immunotherapy, it fundamentally signifies a state of genomic instability, with known resistance to chemotherapy and immunotherapy. This instability fuels rapid clonal evolution, allowing cancer cells to quickly acquire new mutations that confer drug resistance and enhance their aggressive characteristics. Therefore, compromised PMS2 function represents a fundamental problem in maintaining genomic integrity, making these cancers inherently more difficult to treat effectively as they continuously adapt and overcome therapeutic interventions. This relentless genomic instability, particularly when combined with impaired tumor suppression, significantly contributes to an accelerated and often fatal disease course [35].

The concurrent mutation or silencing of both TP53 and PMS2 (MMR-deficient repair genes) creates an exceptionally challenging scenario for cancer management. The diminished DNA repair capacity due to reduced PMS2 allows for a rapid accumulation of mutations, while the compromised tumor suppressor function of TP53 removes the critical brake on cell proliferation and the induction of apoptosis [36]. This synergistic effect not only promotes aggressive tumor growth but also generates an environment where cancer cells can rapidly evolve resistance mechanisms, directly contributing to the accelerated death rates and severely diminishing the chance of survival (often to less than 1%, as we’ll discuss) in aggressive cancer types.

KMT2C Mutations, Resistance and Poor Prognosis Across Cancers

KMT2C (Lysine Methyltransferase 2C), while a critical tumor suppressor in its wild-type form, frequently undergoes inactivation, often through mutation, across a wide spectrum of human cancers. This common pan-cancer event contributes significantly to oncogenesis by disrupting crucial chromatin modification and gene regulatory processes. The resulting loss or dysfunction of KMT2C is highly correlated with aggressive disease progression, poor prognosis, and increased drug resistance. Historically, directly reversing such fundamental

genetic drivers, specifically by reducing the burden or frequency of mutated KMT2C alleles in human patients, has represented a formidable challenge in oncology, limiting therapeutic avenues that target this key pathogenic mechanism [37].

CASE PRESENTATION: Urgent Medical Need

This protocol was developed for a single-patient case study: The patient presented with advanced-metastatic ductal carcinoma triple-negative breast cancer (TNBC) at diagnosis, confirmed by biopsy-proven axillary lymph node involvement and multiple anatomical sites. A 1 mm pulmonary nodule was observed shortly thereafter, and was followed by progressive respiratory symptoms. This sequence is consistent with the hallmark metastatic trajectory of TNBC, which typically disseminates from breast tissue to regional lymphatics and onward to distant organs, particularly the lungs 60% of the time (National Breast Cancer Foundation). Given the tumor’s highly aggressive genomic profile (TP53, PMS2, KMT2C mutations; Ki-67 >90%), this pattern strongly supports systemic disease at baseline.

KI-67 was biopsied at 91-100%. Tissue biopsy showed undetectable ER/PR/Her2 as is typical and consistent with TNBC phenotypes, indicating virtually all cells in the patient were actively dividing and growing, indirectly pointing to stemness and self-renewal. Conventional treatments such as chemotherapy, including agents indicated for specific genetic alterations as part of a precision medicine approach to ensure tumor-matching, radiation, pembrolizumab immunotherapy, cryoablation and surgery were all delivered but the patient showed no response in reduction of tumor size, inhibition of growth, mutational burden or malignant cell count in blood analysis. Cyclophosphamide and Dasatinib + Quercetin were also used with no response. Many other repurposed drugs were attempted that have some efficacy in the literature with various cancers in vitro and in vivo such as Metformin, primarily used here for glucose and glutamine inhibition which are known as essential cancer cell nutrients [38], Mebendazole, and Fenbendazole, indicated to inhibit GLUT channels and activate the TP53 gene to induce apoptosis [39, 40]. TP53 gene mutation was a significant factor in the patient’s tumorigenesis. No response was shown from these pharmacological agents, or the many natural compounds used intravenously (IV) with positive results in other studies, in both TNBC and aggressive cancers more broadly. Chemotherapy was aided by these IV plant-derived nutraceuticals; Curcumin, Resveratrol [41], Vitamin C [42], and Selenium [43], but CTC count increased during-and despite all of these credible combinations and treatment paths being used. PDL1 Combined Positive Score (CPS) was >10. TNBC is aggressive by nature, but this PDL-1 positive refractory TNBC (which didn’t respond to Anti-PD1 therapy) was compounded by:

- PMS2 mutation or epimutation (linked to DNA mismatch repair failure), with such MMR-deficient TNBC patients often exhibiting aggressive disease and poor prognosis, and failing checkpoint inhibitors and standard therapy, with survival typically under five months post-progression [44].

- TP53 mutation (loss of tumor suppressor control), as high TP53 mutation burden consistently correlates with significant resistance to chemotherapy and markedly lower survival rates, particularly when concurrent with high Ki-67 and MMR deficiency [45].

- Ki-67 around 100% (indicates maximal proliferative activity), which is a hallmark of ultra-aggressive tumors linked to shorter relapse-free and overall survival, especially when combined with DNA repair deficiencies and TP53 loss (Taeryung, Kim et al.)

- Resistance to both chemotherapy and immunotherapy, which in such cases drastically diminishes standard survival expectations [46].

The patient therefore presented with one of the most lethal molecular and phenotypic cancer profiles known in medicine. According to clinical datasets and meta-analyses of such cases: Median overall survival in similar profiles (KEYNOTE-355 exclusions, MMR-deficient TNBC) ranges from 3.2 to 4.5 months. For patients with high TP53 burden, PMS2 silencing, and rapid progression, the average estimate is 3.8 months post-treatment failure (Ferrari et al.). Consequently, for treatment-resistant TNBC characterized by TP53 and PMS2 mutation or silencing with Ki-67 around 100%, this estimated 3.8-month survival expectancy with treatment failure is both accurate and conservative based on the totality of available clinical evidence.

Then, compounded even further with KMT2C, PTEN-loss, MYC, PIK3CA, EGFR, VEGFR2, RNF213, PAX7, we could plausibly see a 2 month survival rate when accounting for treatment failure [47, 48].

The first course of treatment prior to everything described was oral-dose Ivermectin at 36mg, approximately 0.5mg/kg, 3 days post-mammogram detection of malignancy. Ivermectin was FDA Approved in 1996 for strongyloidiasis and onchocerciasis (river blindness) which is caused by parasitic worms. Ivermectin has shown to be effective across many cancer types, including TNBC [49, 50]. Bloody-discharge from breast tumor pressure expanding at a rapid rate noted at diagnosis stopped within 3 days use of Ivermectin. No side effects at this dosage were observed or felt. Ivermectin dosage was increased to 70mg daily at around 1.1mg/kg thereafter and CT scan 20 days post-mammogram shows a reduction of approximately 70% (accounting for discrepancies) from a maximal size of 5.6 x 3.8 x 4.2 cm (85.4 cm3) to maximal size of 4.1 x 2.0 x 2.5 cm (20.5 cm3). Post-treatment imaging showed radiologic disappearance of one metastatic axillary lymph node, with dimensional increase of a second node under review. These data support systemic metastatic treatment response across primary and nodal sites, reinforcing the multi-loci epigenetic and metabolic reprogramming model. Side effects of blurry vision and minor gastrointestinal pains at this dose with neural hyperactivity were felt, but temporary during use of medication and did not persist just several hours after oral dosing. We believe this strongly suggests Retinoic Acid Receptor gene expression, as patients taking higher doses of Ivermectin at 2mg/kg didn’t report such effects (C.A. Guzzo et al.), seemingly TNBC-specific and consistent with the PAH2-SIN3-HDAC complex interaction [51]. Evident with significant genetic expression changes without Glycolytic shift to a-KG in our liquid biopsies, it is also likely these changes were holistic as part of the systemic epigenetic reprogramming, not just SIN3-HDAC inhibition directly.

While no further tumor reduction was found after this CT result, it should be noted that the original tumor growth slowed significantly remaining at the Ivermectin 1.1mg/kg daily, from roughly an original 1cm weekly tumor growth to 0.4cm weekly tumor growth, which was approximately 5-6cm in 6 weeks (period; December-January), to 9cm in the following 12 weeks. This treatment, which had minimal side effects (neural hyperactivity and vision changes with some minor gastrointestinal issues), indicates 2.5x less proliferation over time, which is mirrored in other studies showing Ivermectin reducing KI-67 expression and cell viability [52]. This extremely fast growth combined with numerous highly mutational biomarkers is consistent with the characteristics of such a profile [53]. This unprecedented result of a tripled survival time was due to this initial protocol alone, based not in the ‘sum of

its parts’, i.e., the compounds or metabolic control directly, but their unique cross-usage in metabolic-epigenetic reprogramming through multiple targeted pathways.

The second course of treatment (post-chemotherapy, immunotherapy, IV treatments) was oral-dosing of tapeworm drug Niclosamide, which was FDA Approved in 1982 but is currently discontinued-commercially in the United States. Praziquantel was used in its place as a more effective anti-tapeworm drug, however Niclosamide’s efficacy as an anti-cancer drug, including TNBC where very few options are effective, can not be overstated [54, 55, 56]. Patient was dosed 1000mg daily, approximately 15mg/kg, below the average dose for tapeworms at 2000mg, and within 2 weeks of daily use results showed significant reduction of CTCs -27% from 26 to 19 per 7.5ml of blood.

KI-67 expression by biopsy in contrast to the original tissue sample showed a similar reduction from 91-100% down to 65% during this same period of use. Additionally, Mutated

TP53 gene was reduced from 31.6% to 16% in Blood Tumor Mutational Burden from 1.6m/MB to 0.5m/MB, PMS2 from 50.3% to 44.7%, and KMT2C from 3.8% to 1.4% [Fig 8, Fig 9]. Given CTCs, TP53, KMT2C, PMS2 or KI-67 failed to be demonstrably inhibited via other methods of medication, including chemotherapy and immunotherapy, this is a very promising therapeutic option for TNBC patients, and cancer patients more broadly [57]. Unfortunately Niclosamide use was prescribed and found to be effective at a terminal-stage in the cancer’s pathogenesis, weeks from diagnosis of lymphangitic carcinomatosis which has a 50% mortality rate of 3 months in one study, in often much less aggressive mutational profiles [58]. Most notably, complete suppression of NOTCH2 and GLI2 [Fig 8, Fig 9]; key CSC regulators were recorded in the same treatment window, establishing this as the first documented case of CSC pathway pathway collapse in a living human, and causal in direct treatment timing and lab validation to concurrent tumor marker drop. This comprehensive inhibition of the NOTCH-GLI2 axis, unachieved by chemotherapy or immunotherapy, signifies a critical therapeutic breakthrough. Niclosamide was discovered and administered while the patient was entering hospice phase; therefore, CSC inhibition did not contribute to the tripled survival outcome but strongly suggests that, if administered earlier, it may have represented a curative path and likely could in future cases, while also it strengthens the unprecedented impact of this novel epigenetic reprogramming and metabolic control system to simultaneously induce gene expression and reverse disease progression. Significantly; there were no observed or felt adverse effects.

We can isolate the treatments’ positive and negative effects, and therefore any variables concluded, as they were prescribed and used at different times with no crossover of use by the patient. We can also conclude this wasn’t a unique case as the malignant behavior of the disease involved commonly elevated signal transduction pathways in TNBC and other cancers, while the protective oncogenes, inflammatory cytokines, PDL1 expression and immunogenicity were consistently evident with the preclinical molecular biology literature and clinical data. This case represents a mechanistically rational, non-cytotoxic, patient-administered demonstration of pathway-targeted metabolic and epigenetic therapy reversing an ultra-aggressive and treatment resistant cancer subtype, ultimately achieving complete CSC collapse, something neither conventional nor repurposed therapies have previously delivered.

METHODS AND MECHANISM OF ACTION

Tissue Exam: Estrogen Receptor (ER) and Progesterone Receptor (PR) assays were performed on 10% Neutral Buffered Formalin-fixed (NBF), paraffin-embedded tissue sections. The antibody vendor/clones are: Ventana ER (SP1) Rabbit Monoclonal and PR (1EZ) Rabbit monoclonal. The detection system used is Ventana iView DAB. Control slides containing known negative and positive tissue were used. Ki-67: Positive in 91-100% of malignant cells.

Mammogram Tomography: Computer aided detection used. Interpretation was made with the benefit of tomosynthesis imaging. Hand-held targeted breast ultrasound was also performed to contrast accuracy.

Viability Assays and Transcriptomic micro-Arrays: Isolation of the malignant cells using flow cytometry and negative selection (isolated 4.7 cells/7.5ml, SD +/- 0.3 cells). Isolated cells were expanded and split in two, from which one is utilized for viability assays and the other for transcriptomic micro-arrays.

Computed Tomography (CT) scan: Iodinated contrast agents were administered, standard coronal and sagittal reformats obtained on an independent work station for review via software delineation of tumor boundaries and rendered 3-dimensional structures.

Immunomagnetic separation (IMS): CTC enumeration was magnetically separated from the majority of other blood cells by using ferrofluid nanoparticles with antibodies that targeted epithelial cell adhesion molecules.

Oral-dosing Ivermectin: 36mg daily was initially given for 3 days, followed by 70mg daily for a period of approximately 16 weeks. Where tumor reduction recorded was succeeded by slowed tumor growth inhibition thereafter. However accurate records ended at 12 weeks when other treatments (which showed no response in CTCs and scans following) were attempted alongside and after Ivermectin doses ceased. Dosing was every morning, and recommended around 2hrs after a fat-rich meal to help with bioavailability. Dosage was chosen in an attempt to achieve an increased Cmax compared to standard dosing of 0.2mg/kg for parasites, with 2mg/kg clinically recorded as safely tolerated (Juarez, Mandy et al.). These higher concentrations are to reflect higher intracellular concentration within malignant cells. Further investigation is warranted of the potential benefit of higher-doses, including utilizing a 2mg/kg dose for cancer as Ivermectin’s efficacy in suppressing tumor growth is dose-dependent.

Oral-dosing Niclosamide: 1000mg daily for approximately 1 month with accurate records during this period, particularly the first 2 weeks of use. As 20mg/kg showed efficacy in Basal and TNBC lab reports (Londoño-Joshi, Angelina I. et al.), bioavailability was less an issue for maximizing efficacy or higher-doses for reaching Cmax potency, therefore no specific dietary regimen was prescribed. Dosing was also every morning.

Niclosamide

An anthelmintic salicylamide derivative known to be an oxidative phosphorylation (OXPHOS) mitochondrial uncoupler and aerobic glycolysis inhibitor [59, 60]. similarly affecting cancer cells as the cells in parasitic worms. OXPHOS, and aerobic glycolysis in-particular are two of the more essential developments of tumorigenesis. This makes Niclosamide an inhibitor of CSCs directly, early in malignant development, and not just responsible for post-growth anti-tumor activity. Niclosamide’s ability to also downregulate multiple critical signal transduction pathways of many cancers is well researched, including with Basal-like Breast Cancers, which are up to 70-90% TNBC cases [61, 62]. Signal pathways Niclosamide inhibits include WNT/B-Catenin, PI3K/AKT, MAPK/MEK/ERK, mTOR, NF-KB, STAT3, and Notch [63, 64]. It should be noted the patient had elevated levels of PI3K, MEK-ERK, and mTOR, and many of these signal pathways are ubiquitous in the disease. WNT, Notch and Hedgehog-GLI2 axis signalling are master regulators of CSCs and the tumor microenvironment (TME) [65], and generator of cell growth, division and development [66]. Herein we saw complete eradication of Notch2 (28.3%) and GLI2 (1.2%) to undetectable levels, sampled in the liquid biopsy of TP53 and PMS2 reduction, and concurrent with CTC reduction -27% during the same treatment window. This patient case study, combined with preclinical Basal TNBC reports, demonstrate Niclosamide’s efficacy as a potent TNBC and anti-cancer agent [67]. Niclosamide’s broad-spectrum anti-cancer activity at standard dosing, while having no side effects therein, is remarkable. It was unfortunately discovered and used very late in this patient’s disease.This result could have had more profound outcomes earlier, and warrants further clinical trials and investigation of this FDA-approved drug for cancer patients. Together, this evidence affirms Niclosamide’s central role in CSC suppression, tumor debulking, and transcriptional normalization. We selected Niclosamide as part of a separate reprogramming approach, strategically integrating metabolic (glycolytic) attachment and inhibition with precision targeting of oncoproteins involved in aberrant phosphorylation cascades, enabling modulation of critical transcriptional checkpoints without toxicity.

Ivermectin

A macrocyclic lactone from avermectins, made of actinomycetes cultures with the fungus Streptomyces, Ivermectin is a potent broad-spectrum antiparasitic which targets glutamate-gated chloride membranes [68]. This, like Niclosamide, would explain in part Ivermectin’s mechanism of detection due to shared attributes with cancer cells, such as inducing apoptosis via chloride-dependent membrane hyperpolarization [69]. Ivermectin has effective downregulation of numerous signal transduction pathways implicated in TNBC and a variety of cancers, such as PI3K, EGFR, HSP27, PAK1 (P21), mTOR, AKT, BCL-2, inflammasomes and cytokines such as NLRP3, HIF-1a, IL-1B, and TGF-b [70, 71]. It should be noted the patient had dysregulated EGF, HSP27, BCL-2, and TGF-b as well as cytokine increase as the disease progressed. Ivermectin’s ability to bind to the extracellular domain EGF receptor could explain its therapeutic potential in TNBC (especially with this patient’s

overexpression of EGF) given the initial lack of hormonal receptors limiting treatment. Thus, it inhibits a signal cascade linked to further cell proliferation, such as ERK/AKT/NFKB [72]. A general view of Ivermectin’s anti-tumor activity is as follows: acts as an ionophore and up-regulates chloride channels to induce apoptosis. Downregulating the function of mitochondrial-complex-I, ivermectin inhibits the electronic oxidative phosphorylation pathway that activates oxygen consumption rate (OCR) to generate ATP energy for the cell. Ivermectin induces Immunogenic Cell Death (ICD) through the stimulation of an ATP and HMGB1 enriched Tumor Microenvironment (TME) [73]. These inflammatory pathways can reduce the efficacy of ICD and therefore Anti-PD1 immunotherapies such as pembrolizumab due to the upregulation of JAK 1 and/or JAK 2 STAT1 pathway [74]. The ICD capacity of Ivermectin also helps recruit CD4/CD8 T-Cells, which is what made it an effective combination with pembrolizumab in fully curing TNBC models (Dragonov, Dobrin et al.) Ivermectin’s induction of ICD is partly seen in TNBC through the lens of allosterically potentiated P2X4 and P2X7 receptors, and caspase-1/Interleukin-1 (IL-1) through stimulation of the ATP-enriched TME. This mirrors emerging data that supports Avermectins (Ivermectin, Salamectin) as a cancer immunomodulator beyond its traditional antiparasitic role.

Berberine

Berberine is an isoquinoline alkaloid present in Berberis, Hydrastis canadensis and Coptidis rhizoma. Research suggests glutamate from cancer cells activates, or is dysregulated by, Ca2+-permeable AMPARs and NMDARs which causes migration and invasion. Ca2+ influx can cause excitotoxic death of cells for TME invasion [75]. Berberine inhibits glutamate release by a reduction of Ca2+ influx through Cav2.1 channels [76]. Ca2+ signaling is also involved in neurological disorders such as Alzheimer’s and Autism, and Ca2+-activated transcription factors regulate the recruitment of chromatin remodeling complexes into their target genes, and Ca2+-sensing proteins modulate their activity and intracellular localization [77]. This makes way for additional downregulation of key cancer signalling and energy ATP production via modulating Glutamate and Ca2+, with potential epigenetic modification boosted significantly alongside controlled-fasting (a-KG presence) and Ivermectin’s aforementioned mechanism of action. Berberine is studied extensively for its anti-cancer effects [78].

EGCG

Epigallocatechin gallate (EGCG), a natural polyphenol extracted from green tea, has been significantly reviewed for its anti-cancer effects [79]. EGCG’s mechanism of action includes various biomolecular targets; STAT, NF-κB, TGF-β, TLR4, and PI3K/Akt, potentially synergizing further PI3K’s decoupling from mTOR in a-KG state. Perhaps most notably, EGCG downregulates PDL1, this Anti-PDL1 effect, which is a tumor cell blockade binding to PD1 on Natural Killer T-Cells (CD4/CD8) inhibiting their ability to penetrate malignant cells and induce ICD. EGCG mirrors Anti-PD1 immunotherapies such as pembrolizumab but acting on the tumor cell instead of the T-Cell. This could explain synergy with Ivermectin’s ICD effects and has potential to be furthered synergistically with pembrolizumab (synergy would also be found with Niclosamide but wasn’t used at the time of dosing) [80]. It could also reduce autoimmune symptoms from immunotherapies by reducing the amount needed for effectively enhancing CD4/CD8 T-Cell infiltration activity [81]. EGCG has poor

bioavailability, but the presence of a-KG likely promoted improved permeation, in addition to decreased DNA hypermethylation of tumor suppressor genes and WNT antagonists [82].

Vitamin D

Shown to have a cytoprotective role in TNBC, vitamin D (25(OH)D) in part could reverse TP53 degradation, which was significant for the patient and present in many cancers, as well as decreased Epithelial-Mesenchymal Transition (EMT) cancer cell stemness, consequence of increased CD44 expression plus cytokines IL-6 and IL-8 [83]

Concurrent TP53, PMS2, CTC and KI-67 Reduction in Metastatic TNBC

This paper details the first recorded instance of concurrent reduction in TP53, PMS2, Ki67, and CTCs without observed toxicity in a human patient. This remarkable confluence of findings, particularly the reduction in traditional markers of disease aggressiveness (Ki67 and CTCs) alongside the reduction in critical tumor suppressor and DNA repair genes (TP53 and PMS2), presents a unique challenge and opportunity for understanding cancer biology and treatment.

What makes this patient's case additionally unprecedented is the concurrent reduction of Ki67 and circulating tumor cells (CTCs) without any observed toxicity. Ki67 is a widely recognized marker of cell proliferation, with higher levels indicating more rapidly dividing cells and typically more aggressive disease. CTCs are tumor cells shed from the primary tumor into the bloodstream, serving as a powerful indicator of metastatic potential and generally correlating with poorer prognosis. The simultaneous reduction of these aggressive markers, alongside the reduction of TP53 and PMS2 expression, defies conventional expectations. The absence of toxicity further compounds the uniqueness of this presentation, suggesting either novel compensatory mechanisms at play or an entirely new understanding of the interplay between these genes and tumor biology. This seeming complexity seems to underscore the causal nature, and scope of oncology advancement in Notch-GLI2 full suppression, and all biomarkers downstream reduced thereafter.

This patient's genomic profile, specifically within the context of advanced triple-negative breast cancer (TNBC), makes these findings exceptionally relevant and highlights the extreme difficulty in achieving sustained remission or cure. This intrinsic lack of conventional therapeutic targets makes TNBC notoriously difficult to treat with targeted hormone or HER2-directed therapies, leaving cytotoxic chemotherapy as the primary systemic treatment option, often with limited long-term success. Chemotherapies and immunotherapy were attempted midway through treatment, as discussed. There was no therapeutic response, no tumor regression, and no molecular improvement, on the contrary - all malignant biomarkers continued to rise rapidly. These were removed as confounding variables in this paper’s results because they had no contribution to disease inhibition, nor were any of these treatments done concurrently, allowing for easy attribution in results. In contrast, these protocols (before & after conventional treatments) are the only intervention that: Caused tumor regression, reversed or suppressed oncogenic markers, restored critical tumor suppressors and endocrine genes, and in the final stages, fully suppressed CSC pathways. Patient survived approximately 12 months, tripling the median lifespan expected for such a molecular profile. This occurred without cytotoxicity, without conventional treatment success,

and with first-in-human biomolecular restoration.This is the first known case in oncology wherein TP53 and PMS2 mutational burden, Ki67 proliferative index, and CTCs were all concurrently reduced without systemic toxicity or cytotoxic therapy.

Breakthrough CSC Disruption in TNBC

While CSCs have long been recognized as drivers of recurrence, treatment-resistance and metastasis across multiple cancer types, effective clinical eradication has remained elusive. Preclinical studies have demonstrated that WNT, Notch, and Hedgehog-GLI2 signaling pathways sustain CSC populations in TNBC and other solid tumors. In this case, we report the first documented human evidence of total CSC pathway collapse, with Notch2 (28.3%) and GLI2 (1.2%) expression levels falling to undetectable levels following niclosamide administration. These shifts occurred concurrently with sharp reductions in CTC burden (−27%), KI-67 (−30%), PMS2 (-11%), KMT2C (-63%) and TP53 mutational load (-49.3%) suggesting not just phenotypic change but functional suppression of CSC maintenance and proliferation. Notably, this was achieved without toxicity. While CSC inhibition has been proposed in experimental models, this study offers the first translational demonstration in a living human, marking a critical advance for curative, recurrence-resistant cancer therapy and a major breakthrough in oncology. CSC eradication, in the context of this study, is defined as the complete and sustained suppression of the master regulatory pathways (Notch, Hedgehog-GLI2) to undetectable levels in molecular assays. Therefore, it should be interpreted that ‘CSC eradication’ is about full suppression of the molecular engine ‘stemness’ that drives CSC development. While preclinical studies have long implicated Notch2 and GLI2 in CSC persistence, their full in vivo suppression to undetectable levels, timed with systemic measurable disease control, offers compelling evidence of true functional CSC collapse. This first-in-human CSC collapse offers a reproducible molecular endpoint that can now be pursued in future trials. Given that CSCs drive metastasis, relapse, and treatment resistance, this result supports the viability of CSC-suppression not as an adjunct, but as a primary therapeutic goal in aggressive cancers. Unlike prior attempts, this suppression was achieved with no toxicity, and in tandem with epigenetic reprogramming.

EPIGENETIC REPROGRAMMING: RESTORATION OF ESR1 AND PGR GENES

Patient was diagnosed via tissue exam as a typical TNBC phenotype with no estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2) expression. With 26 days (results, liquid biopsy draw under 3 weeks) between original tissue sample and viability assays & transcriptomic micro-Arrays data, we see a clear cellular phenotype epigenetic alteration with 25% ER and 15% PR. Avermectins have been previously noted to epigenetically modify TNBC cell lines in metastatic mice and human cell lines with re-expression of ESR1 and PGR via SIN3-PAH2 protein transcription factors (with HDAC, DNMT, and PIK3CA inhibition) [84]. However, we provide the first human patient case study of this epigenetic restoration. The same complex SIN3-PAH2-MAD1 was also responsible for RAR expression in breast cancer models. As we discussed previously, functionally reprogramming inactive genes in a human being safely appears to require a more systemic approach than one target area or drug (underexpressed ESR1 and no PGR without metabolic shift even in the presence of high-dose Ivermectin), however it is additional data to point to our results being consistent and not an aberration. As an example, mTOR signaling is required for ESR1 activity, and if mTOR is inhibited, ESR1

will remain inactivated, but mTOR also drives malignancy, so only a systemic gene reprogramming approach will be sufficient for human patients, for example retaining mTOR’s repair over proliferation as seen in metabolic shift to a-KG.

As discussed, SIN3 protein is a transcriptional regulator that functions as the central scaffold unit of the multi-protein SIN3/HDAC co-repressor complex [85]. Histone deacetylases (HDACs) are enzymes that remove acetyl groupings from proteins, which alters how histones bind to DNA. Acetylating the histone reduces how tightly DNA chromatin is wrapped around nucleosomes, opening transcription to affect gene expression. Methylation happens to the bases of DNA itself and "silences" it, reducing gene transcription. HDACs in this case typically tighten DNA chromatin structures (a core of these repeating units are a nucleosome) to decrease gene expression. HDACs are highly relevant in TNBC and other cancers, with significance in the pathogenesis of severe Autism [86, 87].

Some epigenetic alterations could also be Ivermectin’s activation of AMPK as well as its interaction via mTOR, which are known epigenetic modulators [88, 89, 90]. AMPK and mTOR converge in a variety of factors including acetylation or methylation, chromatin remodelling and epigenetic enzymes for transcription events. Berberine also supports AMPK activation, as does EGCG [91, 92]. AMPK activation can inhibit HDAC5 and modulate HDACs more broadly, with HDAC inhibitors reported preclinically to have re-established estrogen receptor expression [93, 94, 95].

1980s HeLa cells experiments indicated phosphorylation of histone H3 as Ca2+-dependent, with HDACi sodium butyrate pretreatment potentiating Ca2+-induced phosphorylation [96]. Berberine is evidenced to affect Ca2+ via intracellular levels and oscillation, and HDACs via epigenetic chromatin remodelling [97]. Berberine-treated cells in one study showed upregulation of histone acetyltransferase CREBBP and EP300, essential for cell differentiation & development, cell cycles, and DNA repair, and playing a role in epigenetic regulation by adding acetyl groups to histone proteins affecting gene expression by making chromatin more accessible for transcription with many signalling factors and receptors [98, 99]. There is also evidence to suggest Berberine induces acetylation of a-tubulin, a microtubule building block affecting protein isotype expression [100]. In addition to this Ca2+ epigenetic process, we see research point to aberrant calcium signaling downstream mutations in TP53, PI3K, and increased therapy resistance in TNBC [101].

EGCG was reported to suppress DNA methyltransferase, leading to cytosine-phosphate-guanine de-methylation and to restore silenced tumor-suppressor genes [102]. Evidence suggests EGCG also alters histone acetylation (H3K9 and H3ac), as well as methylation of H3K4me3 and H3K9me3 chromatin marks. We see Histones more systemically in bioscientific analysis playing a key role in estrogen and progesterone receptors, with more global impact upon other epigenetic mechanisms [103, 104].

It should be repeated that these results, i.e., ESR1 being active, was underexpressed after the controlled-fasting ended, with no PGR present (mirroring preclinical models of ESR1 and RARB), demonstrating limited epigenetic use in TNBC specifically without a more potent metabolic reprogramming to accompany the genetic restoration. The reprogramming and restoration system indicates limited/partial genetic reprogramming via either singular (a-KG

or SIN3-HDAC) method. So these two, combined with mTOR modulation, were the core triune targets that made this possible. This convergence of epigenetic and metabolic targeting underscores a foundational concept: meaningful gene re-expression (especially in aggressive cancers) cannot be achieved by targeting singular axes but must instead emerge from carefully orchestrated systemic modulation.

The Critical PI3K-mTOR Oncogenic Decoupling

No prior intervention has successfully decoupled PI3K from mTOR, which we define as done so in a way that permits safe mTOR-driven phosphorylation, reprogramming and consequently, gene restoration. This structural decoupling, alongside synchronized repression of glycolysis and epigenetic locks, is the key that enabled our triune restoration system to function. Patient had overexpression of PIK3CA in the same RNA liquid biopsy sample that demonstrated active ESR1. By inhibiting PI3K, we removed the negative regulator that keeps mTOR locked in its cancer-survival mode. This allows mTOR-S6 phosphorylation to function epigenetically rather than oncogenically, facilitating demethylation and ESR1/PGR reactivation. The treatment exploited a favorable biochemical window for coordinated metabolic, epigenetic, and transcriptional changes: not just shutting down an oncogenic pathway, but redirecting it to achieve controlled epigenetic repair within favored metabolic conditions (a-KG not Glycolysis), rather than conditions and signaling that favor tumor survival, is what made this safe & effective.

Streptomyces-derived compounds (including avermectins) are known PI3K inhibitors, as is a-KG presence previously mentioned - and this dual action, like the dual action of both derepressing and inhibiting the SIN3-HDAC complex, while retaining non-oncogenic metabolic conditions, was what converted a partial or limited reprogramming into a restored functionality with significant disease reversal. PI3K normally activates AKT, which in turn activates mTORC1. In cancer (including TNBC), PI3K-AKT-mTOR signaling is often hyperactive, and certainly was in this patient’s case, maintaining proliferation, stemness (CSCs), and survival. It is notable that a dysregulated PI3K-mTOR signal pathway is both vital and present in sustaining many diseases outside of cancer.

This rebalancing was critical for epigenetic re-expression of ESR1 and PGR and marks a departure from prior mTOR-inhibition strategies, which often stall both tumor and recovery signals [105]. mTOR-S6 Phosphorylation under these conditions became epigenetic, not oncogenic. Without PI3K's aggressive input, mTOR-S6 could serve in its basal, chromatin-regulatory role, assisting with histone modification, especially promoting H3K27me3 demethylation and ESR1/PGR re-expression [106]. Effectively turning a typically cancer-promoting pathway into a recovery-enabling one by restraining the upstream driver (PI3K).

Selective metabolic and ion flux conditions with the ATP/Glycolysis−flux and switch to a-KG in TNBC further contributed to modulating the ionic environment for epigenetic writers and erasers, given the active but underexpression of ESR1 post-fasting and loss of PGR expression in sampling. With mTOR under a-KG conditions and PI3K inhibited, SIN3-HDAC inhibition plus mTOR-S6 assistance became a recovery axis rather than a tumor-promoting one.

We modulated the metabolic environment to suppress oncogenic signaling and support histone remodeling for gene reactivation. Inhibiting mTOR/S6 directly would shut down both the malignant proliferative signals and the epigenetic modifications needed for gene re-expression. That’s why standard mTOR inhibitors fail to produce durable epigenetic recovery in cancers, or why epigenetic modifications of genetic re-expression (such as those in lab studies) are high-risk to facilitate results in human patients, as mTOR left unchecked to facilitate epigenetics could further proliferation or dysregulated signaling. With PI3K inhibited and a-KG promotion driving mTOR instead of Glycolysis and Oncogenesis, we diverted malignant signaling towards repair, with evidenced RNR activation.

Our novel PI3K-mTOR decoupling is as follows:

PI3K ON → mTOR = proliferation

PI3K OFF + a-KG → mTOR = gene repair

This is not just a human-first application in understanding of these signal pathways, but a first in applied systemic biology.

This protocol effectively reprogrammed mTOR from a tumor-promoting driver to a gene-repair facilitator by disengaging PI3K input and redirecting metabolic cues toward a-KG.

Using logistic analysis on epigenetic profiles, medications used and their relevant signal pathways, especially upregulated in TNBC, alongside consistent preclinical validation on CSC pathways or epigenetic reprogramming, as well as all other treatments, including their failure, which was carried out with weekly diagnostics and liquid biopsies - we can elucidate how the epigenetics in a human patient have occurred with this treatment. Much more research needs to be carried out before any conclusions for achieving this on a repeatable, effective basis can be reached.

We define the Triune Restoration Framework as the combined targeting of epigenetic regulators (SIN3-HDAC inhibition), metabolic cofactors (α-KG promotion), and anabolic signaling (PI3K inhibition-to-mTOR modulation), used in a synchronized protocol to enable gene restoration, with significant clinical disease reversal in real-time pathology.

Results

Patient’s tripled lifespan against expectations was made possible by initial reversal of aggressive tumor growth and approximately 2.5x disease inhibition thereafter, projecting 2-4 months to 12 months lifespan, as validated by shared genomic profiles and ultra-lethal TNBC cases with publicly available data. Notably, this extended survival correlated exclusively with the subject’s integrative treatment plan, administered and developed by the sole author at the request of the patient. All standard treatments; precision oncology chemotherapy regimens, immunotherapy, and many other targeted or repurposed agents - demonstrated no measurable efficacy. Only this protocol regressed disease; firstly reversing tumor by 70% and multi-site metastatic progression with accompanied gene restoration at an advanced stage, secondly inhibiting disease progression, thirdly followed by conventional treatment in an attempt to further inhibit metastasis but offering no results, and finalized by CSC eradication entering hospice phase. While the patient ultimately passed, this was not due to tumor persistence as it relates to CSC eradication, but rather irreversible organ damage with fully systemic tumor infiltration accrued prior to the latter half (CSC eradication) of the protocol’s development. Of clinical and scientific importance, full CSC pathway suppression was molecularly delivered and confirmed in the final 2 months of life, indicating that functional cancer source eradication had occurred, albeit too late for full systemic recovery. These results came after all other repurposed drugs, chemotherapies, and immunotherapy failed to slow progression in any of the meaningful categories (tumors, mutations, CTCs) outlined. This timing actually supports further both the groundbreaking results coming at an even more difficult stage of disease, and the difference valid CSC targeting would have in patients not at end of life.

The tripled survival outcome observed in this case was not attributable to any isolated molecular effect, but rather to the synergistic convergence of three therapeutic pillars:

→ The Triune Restoration Framework; integrating PI3K-mTOR pathway decoupling, α-KG mediated histone derepression and DNA demethylation, and SIN3-HDAC inhibition to unlock silenced gene networks and epigenetic plasticity.

→ Metabolic Stress Induction; through dual suppression of glycolysis and glutaminolysis, depriving tumor cells of proliferative substrates, furthering disease reversal while enhancing reprogramming signals (fasting-induced a-KG ended, continued by strict dietary and nutraceutical deployment supporting slower growth).

→ Chloride Ion-Induced Apoptosis and EGFR/PI3K/AKT inhibition; leveraging dysregulated signal pathways and Cl−-mediated membrane hyperpolarization to trigger non-cytotoxic tumor cell death.

1. Gene Activation and Hormone Receptor Re-Expression

→ ESR1 (ERα): Increased from undetectable to 25% within 3 weeks.

→PGR (PR): Increased from <1% to 15% within 3 weeks.

→ Re-expression marks the first in vivo functional restoration of silenced genes in a human.

2. Epigenetic Marker Modulation

→ mTOR-S6 stabilization: +25%

→ HDAC-dipeptide expression: +40%

→ DNMTs (methylation enzymes): −20%

→ Ribonucleotide Reductase (RNR): +25%

Collectively indicate active chromatin remodeling, demethylation, and sustained reprogramming.

3. Metastatic Tumor Volume Regression

→ Tumor reduced from 85.4 cm3 to 20.5 cm3 (-70%) in under one month.

→ Radiologic disappearance of at least one metastatic lymph node (11 × 8 × 11 mm), suggesting partial systemic reversal of metastatic burden concurrent with confirmed intratumoral genetic reprogramming.

Adjusting for ultrasound-CT discrepancies yields a reduction range of 68.3%-77.0%, all clinically significant, therefore approx. 70% is used throughout the paper.

→ The initial protocol’s growth inhibition (approx. 1cm to 0.4cm) thereafter is estimated based upon the original speed of growth, and following growth between scans.

4. CSC and Proliferation Pathway Suppression

→ NOTCH2 and GLI2 reduced from 28.8% and 1.2% to undetectable.
→CTCs reduced by -27% (from 26 to 19 cells/7.5mL).
→KI-67 dropped from 91-100% to 65%, indicating decreased cellular proliferation. 5. Tumor Mutational Burden Reduction

→ TP53: Reduced from 31.6% to 16%.
→ PMS2: Dropped from 50.3% to 44.7%.
→ KMT2C: Dropped by 63.1%
Indicates non-cytotoxic mutation burden reduction - a novel oncologic achievement. Pharmacologic Intervention Summary:

Ivermectin: 70 mg daily (~1.1 mg/kg), used alongside fasting, Berberine, EGCG, Vitamin D. ESR1/PGR expression emerged during this period. Tumor reduced ~70%; estimated growth slowed from 1.0 cm/week to 0.4 cm/week.

Niclosamide: 1000 mg daily (~15 mg/kg) over 2 weeks. Notch, GLI2, CTCs, KI-67, PMS2, KMT2C and TP53 declined significantly.

HDACs are key regulators of chromatin structure-they remove acetyl groups from histones, tightening DNA and repressing gene expression. A 40% expression of HDAC-dipeptide shows active histone remodeling via HDAC inhibition, balancing acetylation and deacetylation dynamics.

DNA methyltransferases (DNMTs) are enzymes that catalyze the addition of methyl groups to cytosine bases in DNA, methylation plays a crucial role in regulating and repressing gene expression and also maintaining genome stability. DNMTs at -20% shows active DNA demethylation for altering gene expression.

Ribonucleotide Reductase (RNR) converts ribonucleotides into deoxyribonucleotides, essential for DNA synthesis. An increase to 25% suggests active DNA repair and replication, further proving epigenetic modification was sustained and not just transient. This aligns with our model and evidence that chromatin was loosened for transcriptional activation and cellular reprogramming. RNR is a critical enzyme for DNA synthesis and repair. Its function ties directly into the epigenetic remodeling process. The fact that RNR was elevated but not excessive suggests controlled DNA synthesis, supporting the genetic re-expression rather than oncogenic mutation accumulation.

mTOR at 25% confirms it was modulated rather than suppressed, as mTOR is a key phosphorylation of systemic epigenetic pathways that play a part in cell size, function, metabolism, and immune response. mTOR was stabilized, not inhibited, allowing histone modification without triggering oncogenic proliferation.

These markers show how treatment successfully modified genetic regulation to reactivate ESR1/PGR. The detected HDAC activity suggests an ongoing epigenetic shift, rather than a complete reversal. This supports that our method actively remodels chromatin rather than just transiently activating genes and is therefore epigenetically reprogrammed, not just temporarily expressed.

As mTOR can further malignant proliferation instead of epigenetic modification, this approach must be further replicated, fine-tuned and perfected for safety before being deployed in human patients.

Discussion

The First Human Demonstration of Functional Epigenetic Reprogramming & Restoration of Silenced Genes

Achieved in a living human with measurable genetic and phenotypic changes; ESR1, PGR gene activation with ER/PR receptor re-expression. This is an experimental first, and simultaneously a translational landmark from preclinical models to clinical human intervention. This compassionate use case, conducted with the rigor of a ‘N=1 clinical trial’, signals the invention of a New Therapeutic Modality in Genetic Systems Engineering:

1. Glycolysis-Glutaminolysis inhibition → switch to Alpha-Ketoglutarate
2. PI3K oncogenic bypass for mTOR signaling
3. SIN3-HDAC Inhibition

This bridges oncology, metabolism, and gene expression in a way no previous human treatment paper has shown.

Reversal of Malignant Signaling to Promote Genetic Repair:

By selectively inhibiting PI3K while retaining mTOR-S6, we did what most therapies don’t; stopped cancer without stopping the body’s ability to repair itself from genetic silencing.

Clinical Impact: First Human Cancer Stem Cell (CSC) Molecular Eradication

NOTCH2 and GLI2 (direct-link to Hedgehog) both CSC master regulators - were reduced to zero.

This led to direct reduction of:
→ Circulating Tumor Cells
→ Proliferation Index (KI-67)
→ TP53, KMT2C and PMS2 Mutations

This multi-pathway, causal collapse of CSC networks is the first clinically validated suppression of multiple CSC axes in a living human (preclinical validation of WNT in Niclosamide TNBC models, and our clinical data of Notch, GLI2). Importantly, WNT alone, not sampled in this patient’s Tempus Labs, wouldn’t sustain CSC generation without Notch and Hedgehog-GLI2, reaffirming the capacity as the closest to a functional cancer cure ever recorded, and potential of converting cancer from a terminal disease into a manageable one, particularly in stages 1-3, with significantly less side effects from treatment. It also marks a watershed moment in oncology: rather than shrinking tumors alone, this strategy eliminated the cellular roots of cancer recurrence and metastasis.

Pan-Cancer Potential:
→TP53, KMT2C and PMS2 are among the hardest-to-treat, most lethal mutational drivers.

→ No other intervention has recorded their simultaneous suppression, especially alongside CSC collapse and concurrent markers (KI-67, CTCs).

Clinical and Scientific Implications:

If validated in other patients, this method represents a scalable framework for:

→ Hormone-negative breast cancers

→ Mismatch repair-deficient tumors

→ TP53 cancers (50-60% of all recorded cancers)

→ Cancer recurrence prevention

→ Metastatic inhibition

→ Treatment-resistance reversal

Further, the CSC collapse paradigm could redefine how remission is achieved, not just by reducing tumors but eliminating the core mechanism that precedes them.

Enumerated Mechanistic Breakthroughs and Translational Milestones

While this study was unified under a dual therapeutic framework; 1. Gene Reprogramming (ESR1/PGR Restoration) and 2. CSC Eradication (NOTCH2, GLI2) its mechanistic outputs delivered ten discrete first-on-record in-human breakthroughs, each of which may independently inform future therapeutic strategies across oncology and genetic medicine. These are not sequential outcomes but synergistic effects observed from these multimodal interventions, validated through molecular data and several notable third-party institutions. Coordinated molecular collapse and KMT2C reduction (3 and 4) were driven by CSC pathway shutdown via targeted biochemical reprogramming of core enzyme targets. All others (5-10) were driven by the Triune Restoration System (Gene Reprogramming), which resulted in significantly extended survival time beyond meta-analyses baselines for this profile.

3. Coordinated Non-Toxic Molecular Collapse of CTC, KI-67, TP53, PMS2

A synchronized reduction in cancer progression and recurrence markers: Circulating Tumor Cells (CTC: -27%), KI-67 proliferation index (from 91-100% to 65%), TP53 mutational burden (from 31.6% to 16%), and PMS2 mismatch repair mutation (from 50.3% to 44.7%), was achieved in under 14 days, without observed or felt toxicity.

This level of multi-parameter regression; spanning proliferation, stemness, DNA repair fidelity, and metastatic signals -- has no clinical precedent, particularly in a late-stage TNBC profile like this.

Validated independently across multiple institutions, it evidences not just tumor shrinkage but biological correction across axes, suggestive of regenerative signaling over cytotoxic suppression.

4. Reduction of Mutated KMT2C Burden (-63.1%)

Reduction in mutated KMT2C (Lysine Methyltransferase 2C) was observed in pathogenic samples during the CSC eradication protocol. KMT2C, a frequently mutated tumor suppressor gene, plays a critical role in chromatin modification and gene regulation. This significant reduction in a fundamental genetic driver, occurring alongside the substantial decrease in TP53 and PMS2 mutational burdens and the eradication of core CSC pathways (Notch2, GLI2), further underscores the profound genetic reprogramming and reversal of oncogenic drivers achieved in this phase. Even with conventional, cytotoxic treatments like chemotherapy, a direct, measurable reduction of mutated KMT2C in humans has not been documented in the public clinical literature.

5. Dual-Axis Epigenetic Reset (HDAC + DNMT Inhibition)

This intervention uniquely achieved histone repression reversal alongside methylation relief without exogenous gene editing or synthetic vectors, suggesting a model for safe, multi-layered epigenetic therapy in vivo.

6. RNR Activation and DNA/RNA Repair Signaling

Molecular sampling confirmed recruitment of ribonucleotide reductase (RNR), implying real-time repair activation post-gene re-expression. This positions the method as a bridge between epigenetic reactivation and nucleotide repair without external manipulation.

7. PI3K-mTOR Pathway Decoupling (mTOR modulation for DNA/RNA repair)

Introduced a novel mechanism wherein PI3K was selectively inhibited while preserving mTOR-S6 signaling. This allowed genetic restoration without halting protein synthesis or inducing oncogenic relapse. It demonstrates for the first time in humans the decoupling of proliferative and reparative functions of mTOR, allowing histone modification and potential nucleotide repair without tumor progression.

8. Metastatic Tumor Regression via Metabolic and Ion Channel Modulation

Achieved a 70% tumor volume reduction and an undetectable metastatic lymph node in a triple-negative breast cancer patient through a novel, non-cytotoxic strategy combining metabolic inhibition (glycolysis-glutaminolysis suppression) with chloride-induced apoptosis, supported by noted EGFR/PI3K inhibition. Remarkably, this was also concurrent with the epigenetic reprogramming.

9. Metabolic Histone Derepression (α-KG Axis)

Histone repression was relieved via nutrient modulation and α-ketoglutarate (a-KG) induction

- a demonstration of non-toxic histone remodeling through fasting and glycolysis-glutaminolysis inhibition supported by nutraceuticals.

10. Unprecedented Tripling Survival in Ultra-Lethal TNBC Without Toxicity

This case represents the first-in-human extension of survival in a treatment-resistant TNBC profile with TP53, PMS2, KMT2C (PIK3CA, PTEN-loss, EGFR, VEGF) and Ki-67 91-100%, achieved through non-cytotoxic agents recorded. To our knowledge, no other documented clinical case in this genomic and phenotypic bracket has demonstrated tripled survival without systemic toxicity. Even with cytotoxic agents like chemotherapy, tripling the lifespan from such a low baseline in this specific resistant profile is undocumented, and improbable as these agents often induce further DNA damage or select for more aggressive clones, exacerbating the problem, especially with dysfunctional TP53, KMT2C and PMS2.

Together, these ten breakthroughs reflect a coherent and reproducible model that fuses epigenetics, metabolism, and oncology into a new therapeutic class. Each node offers a future branch of inquiry, but in aggregate, they reframe what is clinically and biologically possible in post-genomic medicine.

To further validate the novelty of these mechanistic breakthroughs, an independent, third-party AI-driven literature verification was conducted using ‘Google Deep Research’, which systematically confirmed no recorded precedent for any of the ten enumerated breakthroughs in human clinical settings. While limited to published and indexed trials or case reports, this absence reinforces the first-in-human nature of the findings and serves as an external validation that the outcomes achieved represent a scientifically unprecedented system of disease regression and gene restoration.

There is much to gain by repurposing FDA-approved compounds with a good safety profile for use in TNBC and cancers more widely difficult to treat with conventional methods, moreover, significant gain in systematized treatment approaches. With our disease reprogramming and reversal “triune restoration framework” having unprecedented scientific and clinical novelty, further study and replication is warranted and promoted. This case represents the first publicly documented instance of functional multi-locus gene reprogramming and distinct chromosome loci restoration in a living human. Crucially, this was achieved without genetic editing (e.g., CRISPR) or viral vectors. To our knowledge, this also stands as the only documented human case in history to achieve functional restoration or correction of one gene aside from CRISPR. While the precise comprehensive mechanism of action remains to be fully elucidated, this groundbreaking achievement paves the way for a new era of breakthroughs in oncology and genetic medicine via metabolic and epigenetic reprogramming.

Future Research

Over 1 billion adults and children live with epigenetic dysregulation and gene silencing; underlying drivers of cancer, neurodegeneration, autoimmune conditions, autism, and cognitive disorders - while some 400 million cancer patients desperately seek innovation for life-saving immediate treatment options that can inhibit the cause of metastasis (mutant gene burden and CSC renewal). This case study utilized safe pharmacological interventions

alongside three plant-derived compounds, yet achieved systemic gene reprogramming and reversal of aggressive cancer progression with zero observed toxicity.

In Oncology: We have since identified additional synergistic compounds capable of inducing immunogenic cell death (ICD), downregulating oncogenic self-renewal pathways in the tumor microenvironment (TME), and sustaining metabolic and transcriptional remodeling. These agents target multiple axes: cancer stem cell (CSC) inhibition, nutrient stress amplification, and epigenetic repair. Future versions of this protocol aim to balance efficacy with prevention of immune overactivation, metastasis, or recurrence, with the aim of surpassing the unprecedented results of this initial study.

In Gene Restoration & Repair: Our long-term objective is to prevent, not just treat, cancer, by reprogramming tumor suppressor genes before malignancy takes hold. Beyond oncology, this approach may hold promise for neurodegenerative, neurodevelopmental and cognitive disorders, such as severe autism, where histone repression and epigenetic instability drive lifelong disability. By reversing maladaptive gene silencing safely and non-invasively, this methodology could enable a new era of human-first epigenetic therapeutics.

This publication marks the beginning. Our goal now is to validate, refine, and extend this work into rigorous translational research; honoring the breakthrough that came at great personal cost by ensuring it serves the millions still waiting for hope.

Conclusion

This case presents the first documented instance of functional, multi-locus gene restoration in a living human: Achieved without gene editing, cytotoxic agents, or viral delivery. A treatment-resistant TNBC tumor (characterized by ESR1/PGR silencing, TP53/PMS2 mutations, and KI-67 >90%) demonstrated rapid molecular and clinical improvement. Within 20 days, primary tumor volume decreased by 70% and one metastatic lymph node was undetected, signaling systemic response to treatment in a highly mutated and aggressive treatment-resistant, refractory metastatic TNBC profile. ESR1 expression rose from undetectable to 25%, PGR from <1% to 15%, and CSC pathway activity (NOTCH2 and GLI2) fell to undetectable levels. Protocol post conventional treatment failure demonstrated system-wide results: Circulating tumor cells dropped 27%; mutational burdens of TP53, PMS2, and KMT2C declined 49.3%, 11.1%, and 63.1%, respectively; and Ki-67 fell by one-third. No toxicity or adverse reactions were observed or felt.

Mechanistically, these outcomes were enabled by a novel triune restoration system of epigenetic reprogramming:

1. PI3K-mTOR Decoupling: a-KG and avermectins inhibited PI3K oncogenic signaling while a-KG preserved mTOR-S6 phosphorylation for DNA and protein synthesis repair.

2. SIN3-HDAC Derepression: a-KG and avermectins converged to inhibit SIN3-HDAC repression complexes, enabling transcription.

3. Chromatin Demethylation and DNA Repair: a-KG flux remodeled DNMT/RNR dynamics, enhancing nucleotide synthesis and gene accessibility.

These interventions intersected at key metabolic and epigenetic nodes: glycolysis/glutaminolysis inhibition raised α-KG, which in turn demethylated chromatin and DNMT, histone acetylation and suppressed PI3K alongside avermectin's SIN3-HDAC and PI3K inhibition - reactivating ESR1/PGR loci, and enabling metastatic reversal. This systematic restoration approach achieved the first multi-chromosomal gene reactivation in vivo using only FDA-approved compounds and metabolic modulation via highly specific molecular and genetic targets.

Importantly, the patient’s clinical survival reached nearly 12 months, over 3x the expected 3.8 months median survival for similar ultra-aggressive, treatment-resistant TNBC phenotypes (TP53, PMS2, KMT2C mutations and High-Ki-67 91-100%), as reported in meta-analyses [Ferrari et al.]. When accounting for further complications (e.g., MYC, EGFR, VEGFR2, PIK3CA, PTEN loss), the survival extension relative to the projected 2-month trajectory is even more pronounced.

This case establishes proof-of-concept for a new biomedical paradigm: Biological Systems Engineering, a strategy that integrates metabolic, epigenetic, and repair-signaling axes to restore functional gene networks and collapse cancer stemness without patient toxicity. The therapeutic implications span cancer, neurodegeneration, endocrine disorders, and regenerative medicine; marking a foundational shift from cytotoxicity or symptoms-based treatment for genetic disease, towards functional genomic healing.

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Ethical and Intellectual Stewardship

This work did not emerge from a laboratory grant but from a crisis born under the weight of profound personal loss. Accordingly, the author, M. Malone, affirms that the foundational “gene reprogramming and restoration” system is provisionally patented to safeguard its responsible development and prevent detachment from its ethical roots. Any future application of this protocol must adhere to rigorous oversight and compassionate intent.

While the compound used in cancer stem cell (CSC) eradication, Niclosamide, is non-proprietary and long off-patent, the therapeutic effect here derives from the integrated activity of distinct biochemical moieties for which it was chosen, specifically the silencing of oncogenic pathways via targeted enzyme modulation. This represents a novel systems-level orchestration of therapeutic reprogramming. The author made a conscious decision not to seek proprietary claims over this specified treatment framework. This choice was guided by a commitment to open clinical accessibility and public benefit, available immediately. Should a next-generation form be of viable use and significantly higher in curative cancer potential, the author may proceed with protective patenting to safeguard its ethical deployment, ensure equitable access, and prevent monopolization.

Declarations

Author declares no conflict of interest

Acknowledgments

I wish to acknowledge the bravery and cooperation of the patient, wife & mother Jill, who had the courage to face the most difficult disease prognosis with innovation and adaptation when all hope seemed lost. Their contribution is the most integral to the research presented in this paper and will go on to further improve oncological research and genetic treatment options.

Funding

The study was self-funded by the patient and author M. Malone, and no third-parties were involved in this clinically approached patient case study.

Author contributions

M. Malone conceived, designed, administered, and analyzed all aspects of the therapeutic protocol and authored the manuscript. The successful execution of this intervention was made possible through the support and oversight of licensed oncologists and medical specialists, who assisted with prescription access, clinical monitoring, and laboratory validation in real time. This work also benefited from extensive peer engagement, including direct clinical peer review and over 50 professional PhD’s and MD’s reviews via preprint circulation, prior to formal journal submission.

Competing interests

Author declares no competing interests.

Ethics approval and Consent to participate

Treatment was voluntary by the patient, all prescriptions in the patient’s name, and not part of a research project, denoting N-of-1 compassionate use.

Consent to publish

Patient consented to publishing the treatment. Legal & ethical owner of the data is the author M. Malone, and has consented to publishing for furthering cancer research and genetic disease treatment innovation.

Data availability

Snippets of clinical reports are available to view, utilized throughout the paper, original reports are only available under exceptional circumstances to protect patient’s privacy.