Treatment options for HER2 positive cancer types have advanced significantly over recent years, focusing on targeted therapies that inhibit HER2 signaling and more recently using HER2 as the anchor-signal to bring tumor killing moieties or effector immune cells near the cancer cell. In our recent study we compared in vivo efficacy in tumor bearing immune-compromised mice using four different modalities in a proof-of-concept format in a HER2+ ovarian cancer xenograft model, SKOV-3. We then tested a panel of HER2 targeting agents in a broader range of clinically relevant PDX models of breast, ovarian and gastric cancer and correlated these results with different characteristics of the PDX models, such as HER2 expression (by IHC) and molecular phenotype (by WES and RNAseq). The range of modalities that were tested in this study comprised small molecules, antibodies, antibody-drug conjugates (ADC) and mRNA-LNP encoded antibodies. Antitumoral activity was measured as tumor growth inhibition (TGI) based on twice weekly tumor volume measurements. The sensitivity of SKOV-3 was heavily influenced by the modality of the respective drug. Whereas small molecule Lapatinib showed only mild efficacy (TGI 40%), Trastuzumab was moderately active (TGI 57%) compared to Trastuzumab-emtansine (TGI 68%). An mRNA-LNP encoding for trastuzumab was the most efficacious treatment with a TGI of 88%. Despite this, none of the treatments induced complete remission. The ongoing analysis of tumor tissue from these studies suggests an escape mechanism via epithelial-mesenchymal transition (EMT) as Vimentin is upregulated and E-Cadherin is downregulated in tumors after re-growth. The superior activity of the mRNA-encoded trastuzumab compared to the clinically approved therapeutic is likely to be due to the elevated and sustained trastuzumab protein levels in the serum of the tumor bearing animals. Broader screening across 33 PDX models revealed ADCs (trastuzumab-emtansin and trastuzumab-deruxtan) as the most efficacious modalities leading to higher TGI values with prolonged persistence of tumor remission. Two therapeutic antibodies (trastuzumab and pertuzumab) showed similar activity patterns despite their slightly different modes of action. HER2 expression correlated with the sensitivity of the PDX model, although this relationship was not strictly linear. HER2 has provided oncologists a tractable target for some cancer types. However, clinical as well as preclinical data indicate the further untapped potential of HER2 as a therapeutic target. Screening studies like those described are important tools to deconvolute sensitivity and possible mechanisms of resistance in a preclinical setting. This provides opportunities to discover biomarkers to better enable patient selection and to provide a better understanding of HER2 biology, further maximizing its therapeutic potential. Eva Oswald, Philipp Meyer, Loreen Weichert, Roxana Redis, Dan Rocca, Louise Brackenbury, Justin Bryans, Julia B. Schueler. Understanding the mode of action and development of resistance towards HER2 targeting agents in preclinical in vivo models [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 4314.

The manufacturing of therapeutic antibodies requires expensive, complex, and frequently challenging production that keeps the cost of treatment in the clinic high. Alternatively, leveraging novel modalities such as mRNA to encode therapeutics circumvents many of the problems associated with the large-scale production of biologics, and instead relies on in vivo expression of antibodies within patients. Importantly, recent work demonstrates that therapeutic antibodies translated from mRNA in pre-clinical models can be readily detected within hours and persist up to several weeks. Peak levels of circulating mRNA-encoded antibodies are comparable to recombinant protein equivalents and have been shown to be within favorable therapeutic ranges in first-in-human phase I trials. Here we outline an mRNA-LNP based roadmap to encode and deliver therapeutic antibodies, using the standard-of-care anti-HER2 antibody Trastuzumab as a case study, and harness a plethora of pre-clinical models to validate the platform. Characterization of mRNA-expressed Trastuzumab in vitro showed robust translation in producer cell lines, retention of antigen specificity and maintenance of heavy chain-light chain integrity as expected. To evaluate in vivo expression of mRNA encoded Trastuzumab, antibody levels were measured in serum after infusing mRNA-LNPs in translational pharmacology models. Plasma concentrations of secreted mRNA-encoded trastuzumab were benchmarked against circulating levels of infused recombinant antibody equivalents and demonstrated superior pharmacokinetics as well as tolerability at lower doses compared to protein-infused groups. Additionally, bioluminescence imaging was utilized to dissect whole-animal biodistribution and sites of mRNA-translation of ALC-0315 containing LNP formulations used for mRNA antibody delivery. To demonstrate anti-tumor potency, mRNA-encoded trastuzumab was purified from producer cell lines and tested in cytotoxicity assays using human NK cells and HER2-positive tumor cell lines. mRNA-encoded trastuzumab robustly induced antibody-dependent cell cytotoxicity (ADCC) to the same magnitude as recombinant biosimilars. In mouse tumor xenograft models infused with LNPs, mRNA-encoded trastuzumab displayed superior in vivo efficacy at comparatively lower doses relative to recombinant formats by persistently reducing tumor growth and volume as well as enhancing overall survival. Overall, the case study data demonstrate that mRNA-encoded therapeutic antibodies could provide a cost effective, alternate strategy for solid tumor immunotherapy and may unlock an approach to democratize and accelerate patient access to biologics in the clinic. Dan Rocca, Roxana Redis, Phillipp Meyer, Ina Rohleff, Eva Oswald, Sarah L. Martin, Rachel Pooley, Matthew Benson, Namrata Jayanth, Maxim Mashrick, Christian Cobaugh, Michael Shaw, Julia Schueler, Louise S. Brackenbury, Justin Bryans. Developing an mRNA nanomedicine platform to democratise therapeutic antibodies [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 3794.

Abstract The manufacturing of therapeutic antibodies requires expensive production, often challenging purification, lengthy stability optimization and complex protein characterization that despite continual improvement, keeps the cost of treatment in the clinic high. Alternatively, leveraging gene-based approaches such as mRNA to express therapeutics circumvents many of the manufacturing challenges and instead rely on in situ production of antibodies within a patient, improving the developability and cost of sophisticated, disease-modifying antibodies. Crucially, recent work demonstrates that therapeutic antibodies translated in vivo from mRNA can be readily detected within hours following infusion into pre-clinical models and can persist up to several days or weeks. Peak levels of circulating mRNA-encoded antibodies are comparable to infused recombinant equivalents dosed to patients and have been shown to be within favorable therapeutic ranges in phase I trials. Here we outline an mRNA-LNP based platform to encode and deliver therapeutic antibodies in vivo that can overcome costly and challenging manufacturing and that demonstrates both robust PK/PD kinetics and potency. We describe how to effectively encode a standard-of-care anti-HER2 antibody - Trastuzumab - using LNP-encapsulated modified mRNA and validate using in vitro as well as in vivo efficacy models. Initial in vitro characterization of mRNA encoded antibodies demonstrates robust translation in producer cell lines, reproducible expression kinetics, retention of antigen specificity and HC:LC integrity when analyzed by ELISA and WB. To evaluate whether Trastuzumab-encoding mRNA-produced protein is detectable in vivo, antibody levels were measured in mouse serum after IV administration of a single dose of mRNA formulated LNPs. Peak plasma concentrations of mRNA-encoded trastuzumab were benchmarked against circulating recombinant Trastuzumab in parallel groups, and pharmacokinetics interrogated. Of note, the majority of mRNA encoded antibody was likely produced in the liver as shown by bioluminescence imaging. To demonstrate the efficacy of the platform, the potency of mRNA-encoded Trastuzumab isolated from producer cell lines will be assessed using in vitro cytotoxicity assays with human PBMCs and HER2+ tumor lines to dissect ADCC or direct tumor growth inhibition. Additionally, to determine whether mRNA-encoded Trastuzumab is able to retain anti-tumor activity in vivo, a mouse tumor xenograft model will be established and the mRNA antibody-dependent effects on tumor volume, growth and morbidity-free survival will be examined. Overall, the data demonstrate that mRNA-encoded therapeutic antibodies could provide an effective, alternate strategy for solid tumor treatment and may unlock a strategy to deliver lead biologics at reduced cost and with improved developability. Citation Format: Roxana Redis, Dan Rocca, Rachel Pooley, Matthew Benson, Namrata Jayanth, Eva Oswald, Alexander Hale, Louise Brackenbury, Justin Bryans. Developing an mRNA encoded therapeutic antibody platform to simplify manufacturing and reduce time to clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2714.

Clinical correlation of miR-155 and TP53 expression with survival in the lung adenocarcinoma - TCGA dataset when distinguishing between TP53 wild-type and TP53 mutated samples. (A-B) Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in samples that express wild-typeTP53 or harbor TP53 mutations that do not affect TP53 function (A), and in samples expressing mutated TP53 that affects TP53 function (B). (C-D) Kaplan-Meier survival analysis for patients expressing high levels of miR-155 and low levels of TP53 vs. low levels of miR-155 and high levels of TP53 in samples that express wild-type TP53 or harbor TP53 mutations that do not affect TP53 function (C), and in samples expressing mutated TP53 that affects TP53 function (D).

The regulation of microRNA (miRNA) biogenesis, function and degradation involves a range of mechanisms, including interactions with RNA-binding proteins. The potential contribution of regulatory miRNAs to the expression of these RNA interactor proteins that could control other miRNAs expression is still unclear. Here we demonstrate a regulatory circuit involving oncogenic and tumor-suppressor miRNAs and an RNA-binding protein in a chemotherapy-resistant ovarian cancer model. We identified and characterized miR-15a-5p and miR-25-3p as negative regulators of hnRNPA1 expression, which is required for the processing of miR-18a-3p, an inhibitor of the K-RAS oncogene. The inhibition of miR-25-3p and miR-15a-5p decreased the proliferation, motility, invasiveness and angiogenic potential and increased apoptosis when combined with docetaxel. Alteration of this regulatory circuit causes poor overall survival outcome in ovarian cancer patients. These results highlight miR-15a-5p and miR-25-3p as key regulators of miR-18a-3p expression and its downstream target K-RAS, through direct modulation of hnRNPA1 expression. Our results demonstrate the therapeutic potential of inhibiting miR-25-3p and miR-15a-5p and the use of miR-18a-3p/KRAS ratio as a prominent outcome prognostic factor.

Dose-response curves for MEC1 and MEC2 cells treated with fludarabine.

Introduction Spinal Muscular Atrophy (SMA), the second most prevalent autosomal genetic disease affecting infants, is caused by the lack of SMN1, which encodes a neuron functioning vital protein, SMN. Improving exon 7 splicing in the paralogous gene SMN2, also coding for SMN protein, increases protein production efficiency from SMN2 to overcome the genetic deficit in SMN1. Several molecular mechanisms have been investigated to improve SMN2 functional splicing.Areas covered This manuscript will cover two of the three mechanistically distinct available treatment options for SMA, both targeting the SMN2 splicing mechanism. The first therapeutic, nusinersen (Spinraza®, 2017), is an antisense oligonucleotide (ASO) targeting the splicing inhibitory sequence in the intron downstream of exon 7 from SMN2, thus increasing exon 7 inclusion. The second drug is a small molecule, risdiplam (Evrysdi®, 2021), that enhances the binding of splice factors and also promotes exon 7 inclusion. Both therapies, albeit through different mechanisms, increase full-length SMN protein expression.Expert opinion Nusinersen and risdiplam have directly helped SMA patients and families, but they also herald a sea change in drug development for genetic diseases. This piece aims to draw parallels between both development histories; this may help chart the course for future targeted agents.

Supplementary Table S1 Clinical characteristics of two chronic lymphocytic leukemia patient datasets; Supplementary Table S2 Clinical characteristics of two lung cancer patient datasets; Supplementary Table S3 Clinical characteristics of the ALL dataset; Supplementary Table S4 Integrated function and pathway analysis on 248 experimentally validated targets of miR-155; Supplementary Table S5 Univariate and multivariate analyses of survival with patient characteristics and miR-155 and TP53 expression as categorical and continuous variables in different patient cohorts; Supplementary Table S6 Estimate of Cox model and multivariate Cox model, as well as the HR estimated based on the model for miR-155 high and TP53 low vs. miR-155 low and TP53 high.

Clinical correlation of miR-155 expression with survival in leukemia. Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in two CLL cohorts, CLL - NEJM (A) and CLL - Italy (B), and in one ALL cohort, ALL - MDACC (C).

In vivo orthotopic lung cancer model for the role of miR-155 in chemoresistance. (A) Injection and treatment schedule for CDDP (green arrows) and anti-miR negative control (NC) or anti-miR-155 liposomal nanoparticles (red stars) for four different treatment groups: mice that were injected with A549-LVEV cells and untreated (group 1), injected with A549-LVEV cells and treated with anti-miR-NC and CDDP (group 2), injected with A549-155LV cells and treated with anti-miR-NC and CDDP (group 3), and injected with A549-155LV cells and treated with anti-miR-155 and CDDP (group 4). (B) Representative pictures of dissected mice belonging to each of the treatment groups described in panel A of this Figure. Tumor nodules are marked by dotted white circles. (C-D) Graphs of the primary tumor size (C) and aggregate mass of nodules in mediastinum (D) of the four treatment groups mentioned above. (E) In situ hybridization for miR-155 for each of the four treatment groups mentioned above. (F) Immunohistochemical analysis for Ki-67 (proliferation) and CD31 (angiogenesis), as well as the TUNEL assay (apoptosis) and TP53 immunostaining for each of the four treatment groups mentioned above.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

The pervasive role of microRNAs (miRNAs) in cancer pathobiology drives the introduction of new drug development approaches such as miRNA inhibition. In order to advance miRNA-therapeutics, meticulous screening strategies addressing specific tumor targets are needed. Small molecule inhibitors represent an attractive goal for these strategies. In this study, we devised a strategy to screen for small molecule inhibitors that specifically inhibit, directly or indirectly, miR-10b (SMIRs) which is overexpressed in metastatic tumors. We found that the multi-tyrosine kinase inhibitor linifanib could significantly inhibit miR-10b and reverse its oncogenic function in breast cancer and liver cancer both in vitro and in vivo. In addition, we showed that the efficacy of linifanib to inhibit tyrosine kinases was reduced by high miR-10b levels. When the level of miR-10b is high, it can "hijack" the linifanib and reduce its kinase inhibitory effects in cancer resulting in reduced anti-tumor efficacy. In conclusion, our study describes an effective strategy to screen for small molecule inhibitors of miRNAs. We further propose that miR-10b expression levels, due to the newly described "hijacking" effect, may be used as a biomarker to select patients for linifanib treatment.

Clinico-pathological characteristics of the metastatic colorectal carcinoma from the third CRC patient cohort. (XLSX 9 kb)

BACKGROUND:Non-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion.RESULTS:We performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients' overall survival.CONCLUSIONS:The primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific.

Abstract Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors. Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer. Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891–904. ©2016 AACR.

In recent years, understanding the crucial role played by cellular homeostasis in disease initiation and progression became the focus of scientists and clinicians. This SnapShot sketches the involvement of both short microRNAs and long ncRNAs in the major metabolic pathways altered in diseases. To view this SnapShot, open or download the PDF.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.