Abstract Platinum-based chemotherapy remains a cornerstone of treatment for triple-negative breast cancer (TNBC), yet the molecular determinants governing platinum response remain poorly defined. By leveraging the randomized Phase II INFORM trial, which compared neoadjuvant cisplatin to anthracycline-based therapy in BRCA1/2 -mutant breast cancer—we identified miR-362-3p as a specific regulator of cisplatin sensitivity. Higher plasma miR-362-3p expression was exclusively associated with favorable clinical outcome in the cisplatin arm, with no association observed in the AC arm, decoupling platinum-specific vulnerability from general chemotherapy response. We used gain- and loss-of-function TNBC models to establish that miR-362-3p functions as a potent sensitizer to cisplatin in vitro and in vivo . Integrated TCGA analysis and experimental validation identified BCLAF1, a key regulator of DNA damage response, as a direct repression target of miR-362-3p. We uncovered a novel role for the miR-362-3p/ BCLAF1 axis in overcoming platinum resistance in TNBC.

Histone H4 lysine 20 trimethylation (H4K20me3) is a histone modification that is critical in maintaining genome integrity. Dysregulation of H4K20me3 and KMT5C, the major methyltransferase for H4K20me3, occurs commonly in multiple types of cancer but the mechanisms surrounding how they contribute to shaping the epigenomic landscape remains unclear. Here, we show that KMT5C is involved in non-canonical deposition of H4K20me3, independent of H3K9me3, which was previously recognized as a prerequisite for H4K20me3. This novel subtype of H4K20me3 lacks canonical repressive epigenetic signatures and instead overlaps with multiple activating marks. These activating modifications likely contribute to the dynamic changes in transcript levels upon loss of H4K20me3. The mechanism involved in recruiting KMT5C to these loci is independent of HP1, the factor reported to be involved in recruitment of KMT5C to heterochromatin marked with H3K9me3. Instead, biochemical analyses revealed ZNF280C to be a novel interacting partner of KMT5C, with ZNF280C localizing specifically at H3K9me3-/H4K20me3+ sites. Together, these results suggest a novel, non-canonical function of KMT5C-H4K20me3 that protects vulnerable regions of the genome from uncontrolled expression.

Immunotherapy has improved survival across multiple malignancies but remains largely ineffective in solid cancers such as lung, breast, and pancreatic cancer. A key driver of resistance is the immunosuppressive tumor microenvironment (TME). Although numerous mediators of TME immunosuppression have been identified, therapeutic targeting has provided limited clinical benefit. Tumor-derived extracellular vesicles (EVs) have recently emerged as contributors to resistance, yet their mechanisms remain unclear. We developed human non-small cell lung cancer models to investigate EV-mediated immunosuppression. We identified a distinct Golgi-derived EV subpopulation that potently suppress T cell function and tumor infiltration. These EVs express the trans-Golgi network marker TGOLN2, and exhibit minimal levels of canonical EV markers. TGOLN2 overexpression drives this suppressive phenotype. Clinically, elevated TGOLN2 associates with poor survival and correlate with an immunosuppressive TME signature across more than 20 cancer types, including NSCLC. Collectively, this work defines a previously unrecognized mechanism of TGOLN2-driven, EV-mediated immunosuppression.

Abstract The INFORM trial (NCT01670500) was a randomized, two-arm Phase II neoadjuvant study comparing the efficacy of a platinum-based regimen (cisplatin) versus an anthracycline (AC)-based regimen in participants with germline BRCA1/2 mutations and early-stage, HER2-negative breast cancer. MicroRNAs (miRNAs) are emerging as promising non-invasive biomarkers for disease detection and treatment monitoring. The initial aim of this study was to evaluate whether pretreatment plasma miRNA profiles were associated with treatment outcomes in the INFORM trial cohort. Pretreatment plasma samples from 97 INFORM participants (n=53 cisplatin and n=44 AC) were screened for 352 miRNAs commonly implicated in cancer using the qPCR-based ID3EALTM Cancer Panel. Twenty out of 53 participants achieved residual cancer burden (RCB) score of 0 or 1 with cisplatin and 20/44 with AC. Higher plasma miR-362-3p expression was associated with a favorable response to cisplatin (1.7-fold; p<0.01), but not to AC. MiR-362-3p expression in paired pretreatment tumor biopsies (n=79) did not significantly correlate with plasma expression and was not associated with RCB 0/1 (p>0.05). In TCGA, miR-362-3p expression was higher in breast tumors than in adjacent normal tissue, and higher in triple negative breast cancers (TNBCs) compared with hormone receptor-positive tumors (both FDR<0.001), independent of BRCA mutation or Single Base Substitution Signature 3 (SBS3) status. The miR-362-3p findings in INFORM did not replicate in a sister trial, TBCRC 030 (NCT01982448; 11 out of 46 cisplatin-treated patients achieved RCB 0/1, p=0.46). These results suggest that while miR-362-3p is unsuitable as a circulating biomarker for cisplatin response, it may play an important biological role in mediating cisplatin sensitivity in TNBCs, independent of BRCA1/2 mutations. Functional studies demonstrated that miR-362-3p is expressed and secreted by TNBC cell lines, operates as a tumor suppressor, and generally correlates with cisplatin sensitivity. Overexpression of miR-362-3p rendered MDA-MB-231 and CAL-51 cells cisplatin sensitive, while knockdown induced resistance in MDA-MB-436 cells. Ongoing studies are testing whether these effects translate in xenograft models. In silico and functional assays identified BCLAF1, a DNA damage response (DDR) regulator, as a direct target of miR-362-3p. Overexpression of miR-362-3p suppresses BCLAF1 mRNA and protein levels. Our findings demonstrate that miR-362-3p enhances cisplatin responsiveness by targeting BCLAF1. A miR-362-3p-based therapeutic may be useful as a co-agent to enhance tumor responses to platinum-based chemotherapy. Citation Format: Zhaoji Liu, Shizhong Ke, Xiaohui Li, Catherine Wu, Madison M. Uyemura, Brian R. Sardella, Erica S. Massicott, Lin Wang, Emily K. Aronson, Dimitra Karagkouni, Nikolas Kalavros, Ioannis S. Vlachos, Felipe Batalini, Cristina S. Bogsan, Jit Kong Cheong, Lihan Zhou, He Cheng, Phillip Munson, Erica L. Mayer, Judy E. Garber, Stuart J. Schnitt, Nadine M. Tung, Andrea L. Kasinski, Frank J. Slack, Gerburg M. Wulf, Yujing J. Heng. MiR-362-3p enhances platinum response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7903.

Lung cancer remains the leading cause of cancer-related deaths worldwide, largely due to late diagnoses often coinciding with advanced, metastatic stages. Thus, a deeper understanding of the tumor microenvironment (TME) and the molecular mechanisms driving cancer progression and metastasis is critical. Among the many contributors to the TME, extracellular vesicles (EVs) are crucial in facilitating intercellular communication, enabling the transport of biomolecules such as lipids, RNAs, DNA, and proteins. Of the various biomolecules, microRNAs (miRNAs) have emerged as crucial regulators of both tumor cell behavior and the TME. Indeed, our own research has identified that EVs derived from non-small cell lung cancer (NSCLC) cells carry both tumor-suppressive and oncogenic miRNAs, which exert intrinsic effects on tumor cells and extrinsic effects on the TME. However, the mechanisms governing the selective loading of miRNAs into EVs and their role in modulating TME interactions remain unclear. This study aims to investigate EV-miRNA mediated intercellular communication, with a focus on both selective and non-selective enrichment of specific miRNAs within EVs and their implications in NSCLC. Through small RNA sequencing of EV-derived RNAs from NSCLC cells, we identified enrichment of oncogenic miRNAs, including miR-10b, miR-100, and miR-155, which collectively enhanced the invasive potential of bronchial epithelial cells. Functional antagonism of these miRNAs demonstrated their critical role in driving the invasive and migratory phenotypes induced by EVs. Concurrently, our research revealed a marked downregulation of multiple tumor-suppressive miRNAs in NSCLC cell lines, miRNAs that were selectively enriched within EVs. Sequence alignment analysis identified a conserved motif in approximately 60% of these selectively enriched EV-miRNAs. To determine the functional importance of this motif, we introduced point mutations within the motif that resulted in impaired loading of these miRNAs into EVs and their retention within cancer cells, suggesting that the motif is necessary for this active loading process. Ongoing research seeks to identify key players in the EV-miRNA export pathways and explore the differential loading and expression of miRNAs within EVs in cancer, thereby advancing our understanding of these mechanisms and potentially informing future therapeutic strategies for NSCLC. Samira Piltan, Humna Hasan, Ikjot Sohal, Aadya Pandey, Daniel Urdaneta, Nadia Lanman, Sagar Utturkar, Andrea Kasinski. Selective sorting of tumor suppressive and oncogenic miRNAs into extracellular vesicles: Implications for cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1192.

Lung cancer is the leading cause of all cancer-related deaths, with non-small cell lung cancer (NSCLC) making up ∼85% of lung cancer cases. Activating mutations in the epidermal growth factor receptors (EGFR) are associated with NSCLC development, comprising ∼32% of patients. Standard treatment for NSCLC patients involves targeting EGFR mutations with EGFR inhibitors (EGFRi), yet most patients develop resistance to EGFRi over time. Approximately 55% of the mechanisms involved in EGFRi resistance involve EGFR T790M secondary mutations; however, nearly 15% of these mechanisms are unknown. We have previously shown that the loss of KMT5C drives resistance to EGFRi in EGFR-mutant NSCLC cells. Patient data further supports that the KMT5C transcript level is downregulated following resistance. KMT5C is a histone methyltransferase that catalyzes the trimethylation of H4K20 (H4K20me3), a repressive modification historically known for the involvement of heterochromatin formation and maintenance. KMT5C recruitment through H4K20me3 to heterochromatic regions is a well-known mechanism occurring in H3K9me3-dependent manner. Our preliminary data also suggests KMT5C-H4K20me3 is associated with the regulation of gene-rich euchromatin regions; yet the mechanism of recruitment remains unknown. We hypothesize that recruitment of KMT5C to euchromatin is distinct from the mechanism involved in KMT5C recruitment to heterochromatin. Here, we study the interactome of KMT5C using an unbiased proximity labeling technique, BioID, and aim to identify factors that form a novel complex with KMT5C to enable the recruitment of KMT5C to its target genes in euchromatic regions that drive resistance to EGFRi. Alexandra Glaws, Jihye Son, Andrea Kasinski. Noncanonical regulation of genes by KMT5C-H4K20me3 in EGFR inhibitor resistant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2725.

Immunotherapy has significantly improved the survival of patients with various malignancies. However, immunotherapies have largely remained ineffective in tumors that have reduced infiltration of T cells, also called immune-cold tumors. Despite an extensive understanding of factors that mediate poor T cell infiltration, current therapies that target these factors have shown modest clinical benefit, indicating that other factors contribute to this process. One such factor that has been shown to contribute to immunosuppression are extracellular vesicles (EVs). However, due to significant EV heterogeneity, discovery of specific EV subpopulations that are responsible for these immunosuppressive effects has remained a challenge. Overcoming this challenge, we first established relevant lung cancer models to study EV-mediated T cell suppression and then identified a previously uncharacterized population of lung cancer-derived EVs that strongly inhibit the proliferation and activation of T cells. We further identified that this novel T cell-suppressive EV subpopulation originates from the trans-Golgi network and expresses the classic trans-Golgi marker protein, TGN46. In addition, the TGN46+ EV subpopulation lacks conventional EV markers CD81 and CD63. In the immunosuppressive lung cancer model, while total EVs suppress T cells, specifically removing the TGN46+ EV subpopulation ameliorated this effect. Conversely, overexpressing TGN46 in a lung cancer model that had no EV-mediated effect on T cells was sufficient to increase TGN46+ EV release and the ability of the EVs to suppress T cells, underscoring the physiological significance of the TGN46+ EV subpopulation. Analysis of patient data from The Cancer Genome Atlas (TCGA) indicates that high TGN46 expression (both transcript and protein) correlates with worse progression-free survival in lung cancer and overall survival in other cancer types - glioblastoma, lower grade glioma, testicular cancer, and large B-cell lymphoma. Most importantly, tumor-immune association analysis of TCGA data suggests that high tumor TGN46 expression significantly correlates with an immunologically-cold tumor signature, i.e., low infiltration of CD8+ T cells and activated NK cells, and high infiltration of M2 macrophages, across several cancer types, stressing the clinical relevance of our findings. The data elucidates that TGN46+ EVs are a novel factor that potentially contributes to immune-cold tumor microenvironment. How TGN46+ EVs suppress T cells mechanistically and their role in overall tumor microenvironment modulation are currently under investigation, which may inform strategies to enhance immunotherapy effectiveness in immune-cold tumors. Ikjot Singh Sohal, Sydney N. Shaw, Lauren N. Meeks, Andrea L. Kasinski. Golgi-derived extracellular vesicles lack conventional tetraspanins and mediate immune evasion in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 925.

Treatment of triple-negative breast cancer (TNBC) remains highly challenging due to the absence of HER2/hormonal receptors and its resistance to conventional therapies. Although surgery, chemotherapy, and radiotherapy are available treatment options, these broad-spectrum therapies are limited by their non-specific targeting and severe side effects, underscoring the need for more precise therapeutic options. Targeted micro RNA (miRNA) therapeutics have gained significant attention in this regard. In most cancers including TNBC, tumor suppressive miRNAs are often lost, leading to unchecked oncogene expression. One such miRNA, miR-34a which is transcriptionally activated by p53, regulates several oncogenic pathways by down-regulating genes like MET, AXL, and C-MYC. Restoring miR-34a in TNBC is therefore being explored as a potential therapeutic strategy. However, clinical use of miRNAs has been limited by their susceptibility to nucleases, immunogenicity, and lack of targeted delivery methods. To address these limitations, our lab developed a chemically modified, folate-conjugated miRNA-34a (FM-FolamiR-34a) that is highly stable and can be specifically delivered to TNBC cells overexpressing folate receptor alpha (FRα). FM-FolamiR-34a effectively downregulated its targets and demonstrated promising antitumor activity. However, due to its small size (∼14 kDa), FM-FolamiR-34a was subject to rapid renal clearance, limiting its therapeutic availability. Additionally, endosomal entrapment restricted its cytoplasmic abundance, further reducing its mRNA-targeting efficiency. To overcome these challenges, we developed a novel, chemically modified, folate-conjugated divalent miR-34a with enhanced circulation kinetics and improved endosomal escape. This divalent molecule includes two miR-34a duplex copies linked by cleavable deoxythymidine linkers (dT-linkers), which are susceptible to enzymatic cleavage by DNAse II in late endosomes. The dT-linker facilitates miR-34a release from the receptor promoting endosomal escape and increasing cytosolic availability. The dual nature of our construct also raises its molecular weight above the kidney clearance threshold (∼30 kDa), preventing rapid excretion and providing additional time for engagement with FRα-expressing cancer cells. Furthermore, delivering two miR-34a strands per receptor doubles the intracellular miR-34a copy number, enhancing cellular availability. We anticipate that our divalent miR-34a will demonstrate superior antitumor efficacy in TNBC compared to the monovalent version (FM-FolamiR-34a). This innovative approach addresses key critical barriers in miRNA-based therapeutics - specifically endosomal entrapment and rapid renal clearance - presenting an effective strategy to target FRα overexpressing TNBC. Sharjeel Anjum, Shreyas Iyer, Digambar Waiker, Andrea Kasinski. A divalent and chemically modified microRNA-34a with enhanced circulation kinetics and improved endosomal escape for the treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6691.

Osteosarcoma is a common type of bone cancer primarily observed in children, teenagers, and young adults. Current treatments (including surgery, chemotherapy, and radiotherapy) are invasive and induce aggressive side effects. Alternative approaches such as using microRNAs (miRNA) are emerging as promising anticancer agents. miRNA modulates tumorigenesis via tumor-suppressive and oncogenic miRNAs. Tumor-suppressive and oncogenic miRNAs respectively downregulate oncogenic and tumor-suppressive mRNA transcripts. Hence, leveraging the anticancer activity of tumor-suppressive miRNAs appears as a viable anticancer treatment approach. The overarching challenges with miRNA therapy are 1) achieving specific non-toxic delivery of tumor-suppressive miRNA to cancer cells, and 2) enhancing the stability of the miRNA, which can be degraded by nucleases. To circumvent these obstacles, we developed a folate-conjugated fully modified miRNA-34a that specifically targets osteosarcoma cells, which often overexpress folate receptor-α on their surface. The fully modified miRNA-34a bears phosphorothioate linkages at the 3’ and 5’ ends of the sense and antisense strands which significantly reduces nucleases attack. The substitution of the 2’ hydroxyl with 2’ methoxy and 2’ fluoro groups in an alternative fashion further enhances stability. Overall, the fully modified miRNA-34a shows continuous downregulation of target genes 120 hours post-delivery in breast cancer in-vivo models. Here, we present preliminary results for folate-mediated delivery of fully modified miRNA-34a in osteosarcoma samples. This study highlights the potential of miRNA-34a as a safe and effective anticancer agent for osteosarcoma treatment. Iman T. Mevaa, Ahmed M. Abdelaal, Ikjot S. Sohal, Shreyas G. Iyer, Kasireddy Sudarshan, Harish Kothandaraman, Nadia A. Landman, Philip S. Low, Andrea Kasinski. Folate-mediated delivery of tumor-suppressive miRNA-34a to treat osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1864.

A major driver of cancer progression involves the downregulation of tumor-suppressive miRNAs like miR-34a, which regulates key cancer-promoting genes such as MYC, AXL, and BCL-2. Thus, it is not surprising that miR-34a restoration has been proposed as an anticancer strategy. miRNAs, such as miR-34a can precisely target cancer-related genes with limited toxicity, provided they are properly designed. Effective therapeutic design must address RNA specificity, stability, and endosomal escape. Our lab has made significant progress in addressing these unmet challenges, particularly in enhancing specificity and stability of miRNA therapeutics through ligand-mediated delivery and chemical modifications, respectively. Our ligand-mediated delivery strategy capitalizes on overexpression of Folate Receptor 1 (FR1) in cancer cells, and uses the high-affinity FR1 ligand, folate, to deliver miRNAs to cancer cells with high specificity. To overcome the issues with poor instability, we created a fully modified version of miR-34a (FM-34a), increasing its stability by ∼400-fold. Despite significant progress in developing FM- 34a as a therapeutic agent, efficient endosomal escape remains a major hurdle. To address this, a small molecule ionophore, nigericin that disrupts endosomal membranes was conjugated to the folate-FM-34a conjugate (Fol-N-FM-34a). However, nigericin alone achieved only modest improvements in escape efficiency, likely due to the inability of folate to dissociate from the receptor at the elevated endosomal pH levels induced by nigericin. To overcome this limitation, a lower affinity ligand of FR1, 5-methyltetrahydrofolate (5-MTHF), was used in combination with nigericin (5M-N-FM-34a). Unlike folate, 5-MTHF retains a similar binding affinity for FR1 at physiological pH but dissociates more effectively at the higher pH levels attainable in the presence of nigericin, thereby enhancing endosomal escape and cytosolic delivery. We validated that the 5-MTHF ligand binds to FR1-expressing cells in both in vitro and in vivo models and that 5-MTHF-N-FM-34a lead to a higher cytosolic abundance of miR-34a and superior downregulation of miR-34a target genes compared to Fol-N-FM-34a in vitro. To evaluate in vivo activity both single dose targeting and multi-dose efficacy studies were conducted. 5M-N-FM-34a achieved greater target downregulation and enhanced anti-tumor activity in a KRASG12D p53-/- genetically engineered mouse model of non-small cell lung cancer in comparison to other ligands. These findings highlight 5-MTHF-nigericin as a promising ligand for targeting FR1-overexpressing tumors. Kenan E. Ozcan, Sophia A. Matthias, Shreyas G. Iyer, Ahmed M. Abdelaal, Sudarsan R. Kasireddy, Philip S. Low, Andrea L. Kasinski. The combination of 5-methyltetrahydrofolate and nigericin enhances the cytosolic release of chemically modified miRNA-34a, significantly boosting its anti-tumoral activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6692.

KMT5C-mediated histone H4 lysine 20 trimethylation (H4K20me3) has traditionally been linked to heterochromatin formation and maintenance, playing a crucial role in maintaining genome integrity. Emerging evidence, however, indicates that perturbations of KMT5C-H4K20me3 are also implicated in various cancers, positioning KMT5C-H4K20me3 as a promising target for anti-cancer therapies. Despite this, the precise mechanisms underlying KMT5C recruitment to its genomic targets and the specific genes it regulates remain poorly understood. In this review, we explore the dysregulation of KMT5C-mediated H4K20me3 in cancer, providing a comprehensive overview of its known functions. We also highlight recent findings that suggest a novel, non-canonical pathway for H4K20me3 deposition by KMT5C, and, while early on, insight into future opportunities for therapeutic intervention.

Prostate cancer is a leading cause of cancer-related mortality in men. Current therapeutic options include surgical intervention, chemotherapy, immunotherapy, and localized or systemic radiation therapy. However, advanced metastatic prostate cancers are challenging to treat, underscoring the critical need for more effective strategies. Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein, is overexpressed up to 1000-fold in ∼90% of prostate cancers. The high expression level of PSMA, coupled with highly specific targeting ligands, makes it a promising target for diagnostic and therapeutic applications. While various modalities can be delivered via PSMA, delivery of microRNAs has many advantages including the ability to overcome resistance often observed with single-agent treatments due to the ability of miRNAs to downregulate multiple genes simultaneously. Among the miRNAs with anti-cancer properties, miR-34a is among the most promising. Our work and that of others has determined that restoring miR-34a leads to tumor regression. Unfortunately, in vivo delivery challenges remain a bottleneck in advancing miR-34a to the clinic. These challenges can be subclassified into three key issues: 1. achieving specific delivery, 2. avoiding nuclease-mediated degradation, and 3. overcoming poor circulation half-life of the miRNA conjugates. Our lab is dedicated to overcoming these challenges. To address these issues, we developed a miRNA-34a vehicle-free strategy that delivers miR-34a to prostate cancer that comprises three critical attributes. Firstly, a peptidomimetic ligand (PSMA-617) was selected based on its selective specificity for PSMA binding, reducing delivery to non-tumorigenic cells. Secondly, the miR-34a is fully modified (FM-miR34a), containing an alternating pattern of 2′-O-methyl and 2′-fluoro modified sugars and phosphorothioate linkages at the 3′ and 5′ ends of each strand to reduce immunogenicity and to provide exonucleases resistance in-vivo. Thirdly, 4-(p-iodophenyl)butyric acid, a small molecule that exhibits high affinity for serum albumin, was added to the conjugate, enhancing the pharmacokinetic properties. Through binding to serum albumin, systemic circulation of the PSMA-617-miRNA-34a conjugate is prolonged, providing additional time to achieve receptor saturation, leading to increased bioavailability and therapeutic efficacy. Overall, our data indicates that bringing all three of these components together into a holist vehicle can enhance the clinical utility of miR-34a for treating prostate cancer. Digambar Kumar Waiker, Ahmed M. Abdelaal, Shreyas G. Iyer, Ikjot Singh Sohal, Esteban A. Orellana, Kasireddy Sudarshan, Kenan E. Ozcan, Andrea P. dos Santos, Philip S. Low, Andrea L. Kasinski. Ligand-directed delivery of chemically modified miR-34a to prostate cancer: enhanced targeting and extended pharmacokinetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6700.

MicroRNAs (miRNAs) represent a promising class of therapeutics due to their ability to down-regulate multiple genes simultaneously. This offers a significant therapeutic advantage in cancer, where heterogeneity often activates different pathways in different patients. Chemical modifications to the miRNA help overcome challenges associated with nuclease susceptibility, high immunogenicity, and the need for high or repeated dosing to achieve therapeutic effects. The main chemical modifications include changes to the ribose and backbone. Ribose modifications, including 2′-O-methyl and 2′-fluoro, improve nuclease resistance and plasma stability and lower the immunogenicity of the miRNA. Phosphorothioate (PS) backbone modifications increase resistance to nucleases and prolong circulation by enhancing serum protein affinity. Integrating these stabilizing chemical modifications with ligand targeting allows for specific delivery of the chemically modified miRNAs to tumors and metastases, bypassing bulky delivery vehicles and improving penetration into dense tumor architectures. Enhancements to ligand chemistry can also overcome endosomal entrapment. Incorporating many of the modifications discussed in this mini-review, the first fully modified version of miR-34a (FM-miR-34a) was developed, marking a significant milestone as the first fully modified miRNA to demonstrate substantial in vivo activity. Ongoing optimization of the chemical modifications and ligand chemistry, and integrating artificial intelligence into the design process are expected to further extend the potential for delivering on the promise of using these Nobel Prize-winning miRNAs as anti-cancer agents.

MicroRNAs (miRNAs) regulate gene expression by binding to sites within the 3′ untranslated region (UTR) of target mRNAs and exert negative control through mRNA cleavage or translational repression. In cancer, downregulation of tumor-suppressive miRNAs is common, prompting exploration of miRNA restoration strategies for inhibiting tumor growth and suppressing key oncogenic pathways. MicroRNA-34a (miR-34a), known for its tumor-suppressive properties, has advanced to clinical trials as an anti-cancer agent. However, clinical implementation faces challenges such as precise tumor-targeted delivery, susceptibility to degradation in circulation, rapid renal clearance and hindered cytosolic entry due to endosomal entrapment. We tackled a few obstacles by generating a first-in-class fully modified miR-34a (FM-miR-34a) supported by a ligand-mediated vehicle-free delivery platform which exhibited enhanced stability and sustained target gene repression in vitro and in vivo. Prior to the generation of FM-miR-34a, the molecule would have degraded in circulation quickly. However, with FM-miR-34a showing increased stability in serum, we can now aim to increase the circulation half-life of the molecule, which would allow more miR-34a to be biologically available. To achieve this, we incorporated an albumin-binding moiety within the delivery ligand, allowing the molecule to bind to human serum albumin, the most abundant protein in blood. This in turn increases the overall molecular weight of the cancer therapeutic, pushing it over the renal clearance limit and thus increasing its circulation half-life. We also observe a potent combinatorial anti-tumor response when FM-miR-34a is combined with paclitaxel, a standard chemotherapy for breast cancer. Paclitaxel induces mitotic arrest at G2/M and apoptosis at G0 and G1/S phases, making it an ideal partner for synergistic effects with miR-34a. This approach will potentially allow for reduced dosing of paclitaxel and contribute to better therapeutic efficacy, thus improving quality of life of triple negative breast cancer patients. The research, thus, aims to contribute to the development of a robust therapeutic agent utilizing the miR-34a restoration strategy, addressing delivery challenges, and exploring synergies with existing treatments for enhanced efficacy against cancer. Shreyas Ganesan Iyer, Digambar Waiker, Ana C. Betustak, Sharjeel Anjum, Andrea L. Kasinski. Development of a fully modified miR-34a (FM-miR-34a) cancer therapeutic: a translational research study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6699.

Epigenetic dysregulation has recently been recognized as a new hallmark of cancer, with dysregulation of chromatin remodelers estimated to affect 10-20% of cancer. Thus, understanding how epigenetic-based mechanisms contribute to cancer initiation and progression is critical. KMT5C is a histone methyltransferase that catalyzes histone H4 lysine 20 trimethylation (H4K20me3), which has historically been associated with formation and maintenance of constitutive heterochromatin regions. Perturbations in KMT5C-H4K20me3 in cancer has been extensively characterized, including our own work in which we discovered that loss of KMT5C promotes development of resistance to EGFR inhibitor (EGFRi) in EGFR-mutant non-small cell lung cancer (NSCLC) cells. Patient data further supports that the KMT5C transcript level is downregulated following resistance to osimertinib, a third-generation EGFRi. Here, we present data to support a non-canonical role for H4K20me3 where it regulates genes outside of heterochromatin. Notably, deposition of H4K20me3 in these regions occurs without the need for H3K9me3, which was thought to be a prerequisite for H4K20me3 deposition. This uniquely deposited H4K20me3 is associated with pathways such as DNA replication and cell cycle transition. H4K20me3 also co-localizes with activating histone modifications including H3K4me3, requiring additional studies to evaluate how dysregulation of H4K20me3 is involved in transcriptional programming in NSCLC to promote EGFRi resistance. Finally, we identifed the KMT5C interactome, which reveals novel interacting partners of KMT5C, potentially uncovering mediators of KMT5C dysregulation. Taken together, our findings unveil novel roles of KMT5C-H4K20me3 in regulating chromatin architecture and suggest etiological mechanism of KMT5C-driven EGFRi resistance in NSCLC. Jihye Son, Alexandra Glaws, Christina Davidson, Ching-Hua Shih, Paula Vertino, Andrea Kasinski. Decoding role of KMT5C-H4K20me3 in EGFRi resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2719.

Use of tumor-suppressive microRNAs (miRNAs) as anti -cancer agents is hindered by the lack of effective delivery vehicles, entrapment of the miRNA within endocytic compartments, and rapid degradation of miRNA by nucleases. To address these issues, we developed a miRNA delivery strategy that includes (1) a targeting ligand, (2) an endosomal escape agent, nigericin and (3) a chemically modi fi ed miRNA. The delivery ligand, DUPA (2-[3-(1,3-dicarboxy propyl) ureido] pentanedioic acid), was selected based on its speci fi city for prostate-speci fi c membrane antigen (PSMA), a receptor routinely upregulated in prostate cancer - one of the leading causes of cancer death among men. DUPA was conjugated to the tumor suppressive miRNA, miR-34a (DUPA-miR-34a) based on the ability of miR-34a to inhibit prostate cancer cell proliferation. To mediate endosomal escape, nigericin was incorporated into the complex, resulting in DUPA-nigericin-miR-34a. Both DUPA-miR-34a and DUPA-nigericin-miR-34a specifically bound to, and were taken up by, PSMA-expressing cells in vitro and in vivo . And while both DUPA-miR-34a and DUPA-nigericin-miR-34a downregulated miR-34a target genes, only DUPA-nigericin-miR-34a decreased cell proliferation in vitro and delayed tumor growth in vivo. Tumor growth was further reduced using a fully modi fi ed version of miR-34a that has signi fi cantly increased stability.

Prostate cancer (PCa) remains a common cancer with high mortality in men due to its heterogeneity and the emergence of drug resistance. A critical factor contributing to its lethality is the presence of prostate cancer stem cells (PCSCs), which can self-renew, long-term propagate tumors, and mediate treatment resistance. MicroRNA-34a (miR-34a) has shown promise as an anti-PCSC therapeutic by targeting critical molecules involved in cancer stem cell (CSC) survival and functions. Despite extensive efforts, the development of miR-34a therapeutics still faces challenges, including non-specific delivery and delivery-associated toxicity. One emerging delivery approach is ligand-mediated conjugation, aiming to achieve specific delivery of miR-34a to cancer cells, thereby enhancing efficacy while minimizing toxicity. Folate-conjugated miR-34a (folate–miR-34a) has demonstrated promising anti-tumor efficacy in breast and lung cancers by targeting folate receptor α (FOLR1). Here, we first show that miR-34a, a TP53 transcriptional target, is reduced in PCa that harbors TP53 loss or mutations and that miR-34a mimic, when transfected into PCa cells, downregulated multiple miR-34a targets and inhibited cell growth. When exploring the therapeutic potential of folate–miR-34a, we found that folate–miR-34a exhibited impressive inhibitory effects on breast, ovarian, and cervical cancer cells but showed minimal effects on and targeted delivery to PCa cells due to a lack of appreciable expression of FOLR1 in PCa cells. Folate–miR-34a also did not display any apparent effect on PCa cells expressing prostate-specific membrane antigen (PMSA) despite the reported folate’s binding capability to PSMA. These results highlight challenges in the specific delivery of folate–miR-34a to PCa due to a lack of target (receptor) expression. Our study offers novel insights into the challenges and promises within the field and casts light on the development of ligand-conjugated miR-34a therapeutics for PCa.

The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of oligonucleotides, especially RNA, poses a substantial challenge in the face of the harsh conditions encountered intracellularly and in circulation. Therefore, evaluating the stability of oligos in serum is of great significance when developing oligonucleotide therapeutics. This protocol outlines a dependable and reproducible method for preparing oligonucleotide duplexes, coupled with confirmation by gel electrophoresis. Subsequently, the protocol defines a mechanism to assess the stability of the oligo duplexes in serum. This protocol seeks to establish a standardized reference for researchers, enabling them to compare the impact of various modifications on oligo stability and assess the degradation kinetics effectively. Key features • Adaptable for use with small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), and other unmodified and modified oligonucleotides. • Does not necessitate any Biological Safety Level clearance and offers a rapid, cost-effective, and entirely in vitro procedure. • Allows researchers to evaluate multiple modification patterns that, when coupled with targeting activity, allow for selecting the best modification pattern prior to in vivo analysis.

Abstract Like the challenges and skepticism that faced the antibody therapeutics field over a decade ago, RNA therapeutics is facing the same. And, like the antibody therapeutics field, we are beginning to realize the clinical impact of RNA therapeutics amiss these challenges. This is most clearly highlighted with the recent approval of mRNA vaccines and RNAi drugs targeted to the liver. Unfortunately, RNA-based drugs targeted to tumors is lagging behind, even with countless years of work that has revealed the power of using RNAi for treating oncological diseases. Lack of success is attributed to inability to deliver RNAi safely and effectively. A successful delivery agent requires multiple features. First, the agent must deliver the RNA specifically to the intended cells. Second, the agent must have a large therapeutic window, meaning that toxicity, if observed, should occur at doses that are orders of magnitude higher than the therapeutic dose. Third, if delivery of the RNA is by way of a specific ligand and receptor pair, as is the case herein, the RNA must successfully escape the endosome. Simply swelling the endosome is not enough if noncovalent interactions between the ligand and the receptor cannot be disrupted. Fourth, the RNA should include appropriate stabilizing modifications to increase intracellular half-life that will reduce dosing and cost. Through hard work and dedication, we have come up with an inclusive, easily synthesized, intramolecular molecule that achieves all of these essential features. Here, the challenges we face, the hurdles we have overcome, and the barriers that still remain with achieving success in reveling the clinical potential of miRNAs as anti-cancer therapeutics will be presented. Citation Format: Andrea L Kasinski. To the tumor and beyond: Tales of a holistic miRNA delivery vehicle [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR007.