The promise of DNA methyltransferase inhibitors for the treatment of triple negative breast cancer

Daifuku R.

Epigenetics Pharma. 244 Davison Head Dr. Friday Harbor, WA 98250. USA.

Email: [email protected]

OPEN ACCESS

PUBLISHED: 28 February 2025

CITATION Daifuku, R., 2025. The promise of DNA methyltransferase inhibitors for the treatment of triple negative breast cancer. Medical Research Archives, [online] 13(2). https://doi.org/10.18103/mra.v13i2.6276

COPYRIGHT: © 2025 European Society of Medicine. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

DOI: https://doi.org/10.18103/mra.v13i2.6276

ISSN 2375-1924

ABSTRACT

In cancer, loss of gene expression occurs about 10 times more frequently because of hypermethylation than mutations. Hypermethylation can inhibit genes that prevent cancer formation and genes responsible for an antitumor immune response, and is particularly common in triple negative breast cancer (TNBC). The reversibility of these covalent modifications makes them attractive targets for therapeutic intervention. There are two approved DNA methyltransferase (DNMT) inhibitors for the treatment of hematologic malignancies, 5-azacytidine and decitabine. Unfortunately, these two drugs have not yet demonstrated efficacy in the treatment of breast cancer (BC). iMN013 is a nucleoside that has a base identical to decitabine and 5-azacytidine that confers DNMT inhibition, leading to the expression of hypermethylated genes. The sugar is identical to that of gemcitabine, a drug approved for the treatment of metastatic BC that inhibits ribonucleotide reductase, a critical target for TNBC survival. iMN013 is active against BC cell lines in the nM range. The prodrug of iMN013, iMN041 was demonstrated to be an immunotherapeutic in a syngeneic tumor mouse model with significant increases in granzyme B in NK and NKT cells, and in the ratios of CD8-T to regulatory T cells (Treg) and CD4-T to Treg cells while inhibiting myeloid-derived suppressor cells, compared to vehicle controls. Decreased numbers of Treg cells were noted in iMN041 treated animals, differing from decitabine which has been shown to stimulate an increase in Treg cells. A significant decrease in MAGE-A positive tumor cells in iMN041 treated mice suggests that these cells are one of the main targets of the activated immune system. Mage-A is frequently elevated in TNBC. iMN041 was effective in mouse xenograft models of solid tumors, including non-small cell lung, pancreatic, renal and TNBC. In the TNBC model (cell line DU4475), iMN041 demonstrated significant inhibition of tumor growth and improved survival of treated mice compared to vehicle control. iMN041 raises the possibility of treating TNBC patients with a more effective and less toxic regimen that will favorably impact survival by derepressing genes, such as the tumor suppressor TP53, resulting in tumor cell apoptosis, and by stimulating of the own anti-tumor immune response.

Keywords

triple negative breast cancer, DNA methyltransferase inhibitors, iMN013, iMN041, immunotherapy

Introduction

About 1 in 8 U.S. women will develop invasive breast cancer (BC) over the course of her lifetime. An estimated 272,454 new cases of BC in women were diagnosed in 2021 in the U.S. and 42,211 women died in 20221. BC is categorized into three major subtypes based on the presence or absence of molecular markers for estrogen (ER) or progesterone receptors (PR) and human epidermal growth factor 2 (ERBB2): hormone receptor (+)/ERBB2 (-), ERBB2 (+), and triple-negative tumors (TNBC) lacking all 3 standard molecular markers (15% of patients)2. Most new treatment options for metastatic breast cancer (mBC) recently approved by the Food and Drug Administration (FDA) are only effective for ER/PR (+) or ERBB2 (+) metastatic tumors, and the subset of patients with metastatic TNBC (mTNBC) is not sensitive to endocrine therapy or molecular targeted therapy. Chemotherapy remains the main systemic treatment, but the efficacy of conventional postoperative adjuvant chemoradiotherapy is poor3,4. There has been some progress in tailoring drugs and regimens to specific patients with mTNBC, but only 30% of patients can achieve a complete response5. Median overall survival for mTNBC is approximately 1 year vs. approximately 5 years for the other 2 subtypes2. Various therapeutic approaches are under development, such as therapies targeting DNA repair pathways, androgen receptor signaling pathways, and kinases. In addition, immunotherapy has also been demonstrated to improve overall survival and response in TNBC6. Another promising approach under evaluation is the treatment of TNBC patients with a DNA methyltransferase inhibitor (DNMTI). A review of current research on treatment of TNBC with DNMTIs is presented herein, as is the therapeutic promise of a novel DNMTI for the treatment of solid tumors, iMN013, and its prodrug iMN041.

Background on DNA methyltransferase inhibitors

There is increasing interest in the possibility of treating mBC with a DNMTI, particularly mTNBC7,8. DNA methylation is highly dysregulated in cancer with hypomethylation of distal regulatory regions and repetitive elements, and hypermethylation of CpG (cytidine-phosphate-guanine dinucleotide) islands in promoter regions. DNA methyltransferases (DNMTs) are a family of enzymes, the most prevalent of which is DNMT1, with DNMT3a and DNMT3b being barely detectable9. DNMT1 and DNMT3b overexpression can lead to inactivation of gene expression that suppresses tumorigenesis. These genes include (1) tumor suppressor genes such as TP53, (2) genes that promote apoptosis, and suppress metastasis and angiogenesis, (3) DNA repair genes, and (4) genes that express tumor-associated antigens10,11. Since this epigenetic change is reversible, it presents an attractive target for therapeutic intervention. The most effective DNMTIs remain those nucleosides having a 5-azacytosine base, and include the two approved DNMTIs, decitabine and 5-azacytadine (azaC), as well as a recently developed nucleoside, iMN013, and its prodrug iMN04112. Decitabine has been shown to inhibit DNMT1, DNMT3a and DNMT3b7. Both azaC and decitabine are DNMTIs approved in the U.S. for the treatment of myelodysplastic syndrome (MDS)13,14. More recently, decitabine has been approved in the European Union for the treatment of acute myeloid leukemia (AML)15. The benefit of current DNMTIs in solid tumors is not as well established and in a meta-analysis of clinical trials in such patients, DNMTIs were shown to be able to improve clinical outcome, although overall response was limited16. Indeed, clinical trials exploring DNMTI monotherapy in BC have not shown significant benefits to date17.

DNA methyltransferase inhibitors have demonstrated the ability to derepress important genes in breast cancer

TNBC promoter hypermethylation events are more frequent compared with other BC subtypes, suggesting that targeting DNMT may have clinical benefits for TNBC patients18. Decitabine has been shown to induce degradation of DNMT1, DNMT3a, and DNMT3b by TRAF6 through a lysosome-dependent protein-degradation pathway and inhibits tumor growth, particularly in tumors with high DNMT protein levels. In a TNBC patient derived xenograft (PDX) model, tumors responded to decitabine regardless of chemotherapy response. Thus, TNBC patients with high DNMT levels and resistance to standard chemotherapy may still benefit from treatment with a DNMTI7. DNA hypermethylation, primarily mediated by aberrant expression of DNMT1, has been associated with poor survival of TNBC patients. The oncogenic effects of DNMT1 in TNBC include: (1) inhibition of ER expression, (2) promotion of epithelial-mesenchymal transition required for metastasis, (3) induction of cellular autophagy and (4) promotion of the growth of cancer stem cells19. Data suggesting that ER is silenced epigenetically has led to attempts to re-express ER and restore its functionality and hormonal sensitivity to provide a therapeutic target for ER-negative BC. One study demonstrated that treatment of ER-α negative BC cells with decitabine, led to the re-expression of ER mRNA and functional ER proteins by specifically inhibiting DNMT1 expression20. The expression of DNMT1 or DNMT3a was significantly correlated with metastasis in patients with BC, and the expression of DNMT3a and 3b was significantly correlated with advanced clinical stages. Overexpression of DNMT1 or DNMT3a led to promoter hypermethylation and reduced expression of BRCA121. BRCA1 is one of the most common tumor suppressor genes, and plays an important role in DNA repair, replication fork protection, cell cycle regulation, and gene transcription regulation22. DNMT3a-mediated hypermethylation can suppress several critical genes in BC, including SOX223 and HIF-1α24. SOX2 plays a critical role in the migration, proliferation and invasion of BC cells25, while HIF-1α is the regulator of cellular adaptation to hypoxia in rapidly growing tumors26. PKD1 is a serine-threonine kinase expressed in ductal epithelial cells and a negative regulator of actin reorganization processes necessary for cell migration and invasion. Expression is lost during BC progression to an aggressive metastatic phenotype, and mediated by hypermethylation and inactivation of its promoter. In vitro, treatment with a DNMTI has been shown to derepress PKD127. Lysophosphatidic acid receptor 6 (LPAR6) expression is significantly reduced in BC. Decitabine was found to upregulate LPAR6, a possible tumor suppressor, in 98 samples of clinical BC28. LRIG1 (leucine-rich repeat and immunoglobulin-like domain) is a negative regulator of receptor tyrosine kinases and a tumor suppressor. Across BC subtypes, LRIG1 expression is lowest in TNBC subtypes. LRIG1 CpG island methylation is most prominent in TNBC lines and patient samples. Decitabine leads to increased LRIG1 transcript expression in TNBC cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines29.

DNA methyltransferase inhibitors play a role in sensitizing breast cancer cells to other drugs

Numerous examples highlight the association between resistance to specific therapies and changes in DNA methylation patterns. For example, trastuzumab resistance correlates with hypermethylation of the TGFB1 gene30. Similarly, resistance to doxorubicin has been linked with hypermethylation of the MSH2 gene in BC cells31. In a study of 23-genes, a chemoresistance signature was developed from chemoresistant BC and was associated with poor prognosis with multiple chemotherapeutic agents. Of several compounds, decitabine was found to be the compound most likely to target the signature, and in BC cell lines the signature-related chemoresistance could be suppressed by decitabine treatment32. The cytotoxicity of paclitaxel, adriamycin, and 5-fluorouracil was analyzed against human BC cell lines MDA-MB-231 and MCF7. In MDA-MB-231 cells, a synergistic antiproliferative effect was observed with a combination of 10 µM decitabine and these three anticancer drugs, while in MCF7 cells, a semi-additive effect was observed33. In a clonogenic assay, decitabine and taxotere in combination produced a greater loss of clonogenicity than either agent alone in MDA-MB-231 cells34. Decitabine was found to enhance the proapoptotic effect of cisplatin on TNBC cell lines that are less sensitive to cisplatin, indicating the potential for combination therapy in TNBC. Decitabine induced the expression of apoptotic regulators and, among them, NOXA was functionally involved in decitabine-induced apoptosis35.

Gene hypermethylation is important in suppressing an immune reaction to breast cancer cells

In an investigation of integrative expression and methylation of 63 cancer cell lines (breast, colorectal, and ovarian) after treatment with azaC, significant enrichment was found for immunomodulatory pathways in all three cancers (14.4-31.3%) including interferon signaling, antigen processing and presentation, and cytokines or chemokines. Strong upregulation of cancer testis antigens (CTAs) was also observed36. In an orthotopic animal models of TNBC, TNBC-intrinsic MYC impaired T cell infiltration and cytolytic function. In vitro molecular studies showed that MYC transcriptionally upregulated DNMT1, thus repressing the type I IFN response via epigenetic suppression of the stimulator of interferon genes (STING). Decitabine could reverse non-inflamed tumors into inflamed TNBC tumors and combination treatment with PD-1 inhibitor effectively reduced tumor growth in MYC-overexpressing TNBC in vivo37.

DNA methyltransferase inhibitors have shown the potential to enhance the effectiveness of adoptive immunotherapy

The influence of decitabine on the immune response has led to studies on the combination of DNMTIs with immunotherapies, helping to overcome the immune escape or resistance to immunotherapy by affecting both tumor cells and the tumor microenvironment (TME)38. This strategy might also be useful in chimeric antigenic receptor-T (CAR-T) cell based therapies, as myeloid derived suppressor cells (MDSC) induced immune suppressive environment is a major obstacle to the normal functioning of CAR-T cells in BC39. Mechanisms to enhance the CTL (CD8-T) response include the upregulation of tumor antigen expression, increased MHC class I expression, and blunting of MDSC expansion. In a study investigating the effect of combining decitabine, with adoptive CTL immunotherapy in the murine 4T1 mammary carcinoma model, expression of ERBB2, MHC class I molecules, and several CTAs was increased by decitabine treatment of 4T1 cells in vitro. Decitabine-treated 4T1 cells stimulated greater interferon-γ release from tumor-sensitized lymphocytes, implying increased immunogenicity. Decitabine treatment improved the efficacy of adoptive CTL immunotherapy in mice with established 4T1 tumors, with greater inhibition of tumor growth and an increased cure rate40. Another study confirmed the expression of the CTA NY-ESO-1 by decitabine. Antigen expression in the MCF7 cell line was significantly increased after three days of decitabine treatment. The efficient recognition of decitabine treated BC cells by the HLA-A2 restricted NY-ESO-1 specific CAR-T cells confirmed that the de novo synthesized NY-ESO-1 antigen is functionally processed and presented41.

Summary of iMN013 in vitro data

iMN013 (previously NUC013) is unique in being both a DNMTI and a ribonucleotide reductase inhibitor (RNRI). iMN013 is comprised of the base of decitabine and 5-azaC, and the difluorinated sugar of gemcitabine. This sugar results in the inhibition of ribonucleotide reductase (RNR). RNR is an enzyme that converts ribonucleoside diphosphate to deoxyribonucleoside diphosphate. In a custom siRNA screen used to identify targets that were critical for growth of TNBC cells, RNR1 and RNR2 were found to be targets for TNBC cell survival42. An effect of RNR inhibition is to decrease the endogenous pools of deoxyribonucleotides, favoring the enzymatic uptake of the therapeutic deoxyribonucleotide, a phenomenon which has been described as self-potentiation43. Gemcitabine is the most widely used RNR inhibitor and is approved in combination with paclitaxel for the first-line treatment of patients with mBC44. As will be detailed below, combination of DNMT inhibition with RNR inhibition results in a unique therapeutic profile for iMN013 that differs in significant ways from both decitabine and gemcitabine. The tumor suppressor gene TP53 is of vital importance in preventing human cancer development and progression. The functions and stability of the p53 protein are often abrogated via posttranslational mechanisms, such as DNA methylation, in the human cancers that harbor TP53 wild type (WT). In the Carolina Breast Cancer Study which analyzed 496 cases of invasive BC, compared with luminal A, basal-like tumors had more TP53 mutations (44% vs 15%, P < 0.001), suggesting that the majority of invasive BC tumors are TP53 WT even if TNBC45. p53 is often inactivated in cancer because it can trigger cell growth arrest, apoptosis, autophagy or senescence, which are detrimental to cancer cells, and it impedes cell migration, metabolism or angiogenesis, which are favorable to cancer cell progression and metastasis46. iMN013 has been shown to inhibit the growth of at least one tumor cell line from all solid tumor tissues tested in the NCI 60 cell line panel, including breast, colon, central nervous system, renal, lung, melanoma, ovarian and prostate. In this panel, iMN013 was more active than decitabine against p53-null/mutant cancer cell lines (p = 0.027) but even more so against p53 WT cell lines (p = 0.0025). This difference in activity is likely mediated by inhibition of p53R2 by iMN013. p53R2 is the p53 inducible subunit of RNR43. Experimental data have shown that decitabine unexpectedly induced more apoptosis in TP53 null cells than in TP53 WT cells47. These data are compatible with a clinical trial of decitabine and carboplatin where of 9 of the BC patients had available TP53 sequencing results. The 6 patients with TP53 mutations had higher objective response rate (3/6 vs. 0/3) and better overall survival (16.0 vs. 4.0 months) than the patients with TP53 WT48. iMN013 has been tested for activity in multiple cell lines. The IC50 of iMN013 in murine mammary carcinoma cell lines EMT-6 and 4T1, as well as human BC cell lines DU4475 and MCF-7, is in the nM range. The DU4475 cell line originates from a patient with TNBC49.

iMN041 as an immunotherapeutic

Because of the lack of therapeutic options, there is considerable interest in immunotherapy for TNBC. Compared to BC, TNBC exhibits a greater presence of infiltrating lymphocytes, thereby establishing a favorable immune microenvironment. TNBC also demonstrates a relatively substantial tumor mutation load, providing an antigenic foundation for immune cell recognition50. Like all nucleosides with 5-aza-cytosine bases, iMN013 is subject to hydrolytic cleavage and deamination. To improve the pharmacokinetics and pharmacodynamics of iMN013 a prodrug was developed, iMN041 (previously NUC041). Based on the observation of tumor inflammation and ulceration in mice implanted with the NSCLC cancer cell line NCI-H460 treated with iMN041, it was hypothesized that the treated nude mice demonstrated enhanced NK cell activity51. To further characterize the immunomodulatory effects of iMN041, iMN041 was tested in a Renca syngeneic model to determine the antitumor immune response in immunocompetent mice. When the tumor reached an approximate volume of 270 mm3, treatment of BALB/c female mice was initiated with 1 mg iMN041 SC every other day for two doses or the same volume of saline. Tumors were harvested and the cell suspension was stained with the appropriate fluorophore-conjugated monoclonal antibodies of surface markers and processed using a flow cytometer49.

Efficacy of iMN041 in a mouse xenograft model of triple negative breast cancer

In an animal model of colon cancer (LoVo), iMN013 demonstrated significant suppression of tumor growth and a survival benefit compared to saline controls and decitabine43. iMN041 demonstrated suppression of tumor growth, and where possible based on the number of events, survival, compared to saline control animals in models of non-small cell lung cancer (NSCLC) (NCI-H460)51, pancreatic cancer (SW1990 and CFPAC), renal cancer (786-O and Caki-1) and TNBC (DU4475)49. In the case of DU4475, BALB/c nude female mice were inoculated subcutaneously (SC) in the right flank with tumor cells (5 X 106) in 200 µL PBS mixed with Matrigel (50:50) for tumor development. The animals were randomized and treatment was initiated when the average tumor size reached 60 mm3. All groups were comparable at baseline. After initiation of treatment, animals were monitored daily for morbidity and mortality. Mice were assigned to one of three groups: 0.6 mg iMN041 SC every other day, 0.8 mg iMN041 SC every other day and saline control SC every other day (n = 10/group). The treated mice had their dose held if the weight loss was > 10%. Treated mice implanted with DU4475 were initially administered iMN041 at these two different doses but mice, particularly in the 0.8 mg group, required dose withholding and adjustments; hence, the dose was decreased in both treated groups to 0.5 mg every other day on study day 8 because of concern that weight loss and drug withholding in the treated groups would complicate the assessment of drug efficacy. Tumor volume in treated mice was significantly smaller than that of control mice: In the 0.8 mg group, on days 4-20 (p < 0.05) and in the 0.6 mg group, on days 4-20 (p < 0.01). The median survival of 0.8 mg treated mice vs. control mice was 21 vs. 18 days (p = 0.019) and for the 0.6 mg treated mice was 25 vs. 18 days (p = 0.0032)49. The DU4475 xenograft is a model of aggressive TNBC and there are few reports in the literature of successful treatment79,80. Despite issues related to optimal dosing, iMN041 significantly inhibited tumor growth and improved survival. For reasons that remain undetermined mice inoculated with DU4475 were unable to tolerate the 0.8 mg every other day dose that was successfully administered in murine xenografts of renal or pancreatic cancer. Mice treated with the lower 0.6 mg dose had better outcomes because they were more likely to be treated on schedule. These data suggest that if the study had been initiated at a dose of 0.5 mg every other day, further improvements in outcome would have been observed.

Conclusions

Treatment of TNBC with a DNMTI is of interest because of the poor outcomes with current treatment regimens. Hypermethylation affects many aspects of cancer development including gene expression, in particular the expression of tumor suppressor genes, and immune response. While effective for the treatment of hematologic malignancies, decitabine and azaC have had limited success for the treatment of solid tumors, including BC. iMN013 combines the DNMTI activity of the 5-azacytosine base with the RNRI activity of gemcitabine into a novel molecule. The prodrug of iMN013, iMN041, has shown activity in multiple solid tumor models, including a model of TNBC. Decitabine has been shown to be more active against TP null cells than TP53 WT, while iMN013 has been shown to be globally more active than decitabine, but particularly so against TP53 WT tumors. The preponderance of evidence is that decitabine stimulates an increase in Treg cells, while iMN041 inhibits the proliferation of Treg. Treg cells mediate an immunosuppressive environment in tumors. Treatment with iMN041 also results in depletion of tumor cells expressing MAGE-A, which is commonly expressed in TNBC, suggesting that MAGE-A is a target of an immune system activated by iMN041. Hence, iMN041, acting through multiple pathways, may be a promising addition to the treatment armamentarium against TNBC.

Conflict of Interest:

The author is the inventor and patent holder of iMN013 and iMN041.

Funding Statement:

None.

Acknowledgements:

None.

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