The Eph receptor tyrosine kinase EphA3 is over-expressed in a range of tumours, most commonly in the tumour microenvironment (TME), including in immunosuppressive cancer-associated fibroblasts (CAFs). In contrast, normal homeostatic tissues show little or no expression of EphA3, making it an attractive therapeutic target. We previously developed a humanized EphA3 antibody (Ifabotuzumab) which was tested in preclinical models and in phase I clinical trials, showing tumour specificity and biological activity. To improve its efficacy, we have tested novel antibody-drug conjugates (ADCs) of Ifabotuzumab with the microtubule disruptor monomethyl auristatin E (MMAE), the Topoisomerase I inhibitor deruxtecan (DXd), and the DNA binding cytotoxin pyrrolobenzodiazepine dimer (PBD), using cleavable linkers and site-specific 'AJICAP' peptide-mediated conjugation. In vitro analysis showed retention of specific binding of Ifabotuzumab-ADCs to EphA3 and specific, dose-dependent killing of EphA3-expressing cells. Ifabotuzumab-PBD showed the highest in-vitro potency and was alone effective against EphA3-expressing cancer-associated fibroblasts. Treatment of mice bearing EphA3-expressing tumour xenografts with all Ifabotuzumab-ADCs decreased tumour growth and increased survival, again with Ifabotuzumab-PBD the most efficacious. Ifabotuzumab-PBD was also able to inhibit growth of tumours that express EphA3 only in stromal cells in the TME, using syngeneic tumour models in immunocompetent mice. Notably, combination with the immune-checkpoint inhibitor anti-PD1 increased CD8+ T cells in tumours, and improved tumour inhibition, compared to anti-PD1 alone. This data indicates that Ifabotuzumab-ADCs have potential as novel specific anti-tumour agents capable of targeting the TME in solid tumours.
PURPOSE Human endogenous retrovirus K (HERV-K/HML-2) is a human-specific endogenous retrovirus family that maintains intact open reading frames and is aberrantly expressed in multiple cancers. We tested whether HERV-K, specifically its envelope (Env) protein, marks clinically relevant diffuse large B-cell lymphoma (DLBCL) states and serves as a tractable immunotherapeutic target. EXPERIMENTAL DESIGN Locus-level transposable element (TE) and HERV expression was quantified in two independent DLBCL RNA-seq cohorts using curated TE annotation and a uniform alignment/counting workflow. Env transcript and protein expression (including cell-surface localization) was validated in lymphoma cell lines using RT-qPCR, immunoblotting, and flow cytometry. Functional relevance was assessed by siRNA-mediated Env knockdown. Candidate extracellular linear epitopes were predicted in silico and synthesized for serological and phage display assays. Anti-Env camelid VHHs were isolated, humanized, and tested for binding, antibody-dependent cellular cytotoxicity (ADCC), and antigen-dependent internalization. RESULTS TE transcripts stratified DLBCL into clinically distinct subgroups, and high HERV-K expression associated with an interferon/viral-mimicry program. Env RNA was detected in patient tumors and cell lines, and the protein was detected on the surface of a subset of lymphoma cell lines, in which Env knockdown reduced proliferation. A humanized heavy-chain antibody (FF-01) bound surface Env, induced selective ADCC in Env-positive models (reporter and primary NK-cell assays), and underwent rapid antigen-dependent internalization. CONCLUSIONS HERV-K activity identifies biologically meaningful DLBCL states and exposes a targetable surface antigen. FF-01 provides proof of principle for antibody-based strategies against HERV-K Env, including ADCC- and ADC-oriented approaches.
Abstract Inactivating mutations in TP53 and ATRX are key alterations defining the genomic landscape of uterine leiomyosarcoma (uLMS), creating chronic replication stress and increasing dependency on Ataxia telangiectasia and Rad3-related kinase (ATR) signaling for survival. ATR-CHK1 signaling limits the efficacy of standard-of-care gemcitabine by facilitating the stabilization of stalled replication forks and preventing lethal DNA strand breaks in checkpoint-deficient cells. Here, we show that ATR inhibitors (elimusertib, ceralasertib, berzosertib) preferentially synergize with gemcitabine over docetaxel or doxorubicin, across a panel of TP53-mutant/ATRX-wildtype uLMS models. When simultaneously administered, gemcitabine/elimusertib demonstrated enhanced synergy and efficacy compared to standard-of-care gemcitabine/docetaxel. This combination benefit was absent in a TP53-wildtype uterine sarcoma control line, highlighting TP53 mutation status as a critical predictive biomarker. Combined gemcitabine/ATRi treatment demonstrated reciprocal potency enhancement at low-nanomolar concentrations and significantly increased apoptotic populations compared to monotherapy. Gemcitabine monotherapy induced CHK1 (S345) phosphorylation and a corresponding S-phase arrest. Elimusertib abrogated this checkpoint, transforming protective S-phase arrest into a cytotoxic response characterized by a marked increase in the sub-G1 population. This transition was driven by the inhibition of gemcitabine-induced CHK1 phosphorylation, resulting in marked accumulation of unrepaired DNA damage (γH2AX) and the induction of apoptosis as evidenced by cleaved caspase-3 expression. These findings demonstrate that elimusertib strongly potentiates gemcitabine-induced damage to kill TP53-mutant uLMS in vitro, outperforming standard gemcitabine/docetaxel when concurrently administered. This combination strategy broadens the clinical utility of ATR inhibitors to uLMS harboring the prevalent TP53-mutant/ATRX-wildtype genotype and supports the clinical evaluation of gemcitabine/ATRi combinations as a novel therapy for uLMS.
Abstract Pancreatic ductal adenocarcinoma (PDAC) displays profound metabolic plasticity that sustains rapid proliferation through heightened demands for nucleotide biosynthesis. Here, we characterize HOSU-53 (JBZ-001), a dihydroorotate dehydrogenase (DHODH) inhibitor demonstrating potent antitumor activity in preclinical PDAC models and define the metabolic determinants of sensitivity and resistance. In HOSU-53-sensitive PDAC cells, DHODH inhibition induces G₂/M cell cycle arrest and triggers apoptotic and ferroptotic cell death. In contrast, resistant cells evade metabolic stress by activating compensatory nucleoside transport and macropinocytosis pathways. Integrated functional and genetic analyses reveal that nucleoside salvage via the equilibrative nucleoside transporter SLC29A1 constitutes a dominant resistance mechanism, enabling uptake of extracellular nucleosides to bypass de novo pyrimidine synthesis. Unbiased metabolomic profiling demonstrates that combined inhibition of DHODH and SLC29A1 precipitates mitochondrial dysfunction, oxidative stress, lipid remodeling, and amino acid depletion, culminating in metabolic collapse and energetic failure. Consistent with these findings, dual inhibition markedly suppresses tumor growth and metastatic burden in xenograft and syngeneic orthotopic PDAC models while reprogramming the tumor microenvironment toward a pro inflammatory, immune activated state. Furthermore, co targeting DHODH and SLC29A1 synergizes with anti–PD 1 immune checkpoint blockade, enhancing immunotherapeutic efficacy. Together, these results establish dual targeting of de novo and salvage nucleotide metabolism as a compelling strategy to overcome metabolic resistance, remodel tumor–immune interactions and potentiate therapeutic responses in PDAC
Abstract Multiple myeloma (MM), the second most common hematologic malignancy, remains incurable despite major therapeutic advances, highlighting an urgent need for novel therapeutic strategies. PCNA is involved in DNA replication and repair, chromatin remodeling, and cell-cycle progression, and increased PCNA expression has been associated with aggressive MM phenotypes and poor clinical outcomes. Cancer cells express a post-translationally modified cancer-associated isoform of PCNA (caPCNA), potentially providing a selective therapeutic target. We evaluated the anti-myeloma activity of the novel caPCNA inhibitor AOH1996 alone and in combination with established anti-myeloma agents in vitro, in vivo, and ex vivo. AOH1996 inhibited growth of multiple MM cell lines in a dose-dependent manner, induced G2-phase accumulation and apoptosis, and reduced sphere formation and colony formation capacity. Combination treatment with bortezomib, venetoclax, melphalan, or cyclophosphamide enhanced anti-myeloma activity relative to single-agent treatment. AOH1996 treatment down-regulated the expression of multiple tumor-associated proteins and inflammatory and pro-survival cytokines. miRNA analysis demonstrated suppression of several oncogenic miRNAs along with upregulation of tumor-suppressive miRNA miR-145. Forced expression of miR-145 inhibited MM cell growth and enhanced sensitivity to AOH1996, implicating miR-145 as a mechanistic mediator of PCNA inhibition’s anti-tumor effects. In xenograft models, AOH1996 significantly enhanced the anti-tumor activity of bortezomib and venetoclax without apparent toxicity. Ex vivo studies demonstrated selective activity against primary MM cells and CD138+ plasma cells with minimal effects on normal PBMCs. Collectively, these findings support caPCNA as a promising therapeutic target in MM and provide strong preclinical rationale for further clinical investigation of AOH1996-based therapeutic strategies.
