Abstract Targeted induction of DNA damage in tumor cells offers a promising strategy to efficiently kill cancer cells while sparing healthy tissues. Precise tumor targeting can be achieved by the exploitation of the differential expression of specific molecular targets on the surface of tumor cells. Somatostatin receptor 2 (SSTR2) has emerged as a valuable tumor-associated target, due to its high expression in gastroenteropancreatic neuroendocrine tumors (GEP-NETs). SSRT2 is not only expressed in these differentiated, slow-proliferating NET, but also in highly proliferative and metastatic neuroendocrine carcinomas, such as small cell lung cancer (SCLC). SSTR2 overexpression occurs in up to 50% of primary tumors from SCLC patients and is correlated with poor prognosis, highlighting SSTR2 as an optimal candidate for targeted therapeutic intervention in a subset of SCLC patients. To exploit this opportunity, we designed a peptide drug conjugate (PDC), named ITF3912 which combines a modified octreotide analog with a novel and potent duocarmicin via a cathepsin-cleavable linker. In preclinical studies in vitro ITF3912 demonstrated high affinity and selectivity for SSTR2, acting as an agonist with an IC50 of 8 nM and a selectivity index of at least 10 over SSTR3/5, with no significant binding observed for SSTR1 or SSTR4. ITF3912 induced cytotoxicity in SSTR2-expressing cells, with efficacy correlated to SSTR2 expression levels. In high level SSTR2 expressing NCI-H524 cells, ITF3912 was rapidly internalized and localized in lysosomes where enzymatic cleavage of the linker released the active toxin. Once in the nucleus, the toxin induced an S-phase replication block and a potent and lethal DNA damage as evidenced by the activation of key biomarkers such as the phosphorylation of ATM, ATR, CHK1, CHK2, RPA32 and γ-H2AX, leading to cell death with an IC50 of 4 nM. The NCI-H69 cell line that expresses lower level of SSTR2 can still be targeted by ITF3912 but with lower efficiency (60 nM). The activity on NCI-H727 cell line that expresses very low level of SSTR2 diminished as expected (400 nM). Consistent with the in vitro findings, ITF3912 showed strong antitumor efficacy in vivo. It effectively reduced the tumor growth of NCI-H524 with a survival rate of 50% after 80 days of treatment and several mice showed complete tumor remission. In contrast, ITF3912 was less effective on NCI-H727 cell line by inducing a tumor growth reduction of 30%. Collectively, these data establish ITF3912 as a potent and selective SSTR2-targeted PDC, whose efficacy in vitro and in vivo correlates with receptor expression levels. These results support the use of ITF3912 as a therapeutic option for patients with SSTR2-positive neuroendocrine tumors, addressing a significant unmet medical need. Encouraging preliminary toxicology results, further support the progression of ITF3912 toward clinical development. Citation Format: Gianluca Fossati, Daniela Modena, Luca Menin, Michela Bottani, Andrea Stevenazzi, Barbara Vergani, Elisabetta Galbiati, Andrea Resovi, Matteo Tironi, Serena Cecchetti, Francesca Spadaro, Maria Carollo, Rosita Lupi, Barbara Valsasina, Paolo Orsini, Silvia Castelli, Italo Beria, Christian Steinkühler. Targeted DNA damage through SSTR2: Preclinical development of a novel peptide-drug conjugate for neuroendocrine tumors [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 1758.

Retinitis pigmentosa (RP) comprises a heterogeneous group of inherited retinal dystrophies characterized by the progressive degeneration of photoreceptors, leading to night blindness and gradual loss of peripheral vision. RP is characterized by a substantial genetic heterogeneity, with more than 85 genes implicated across autosomal dominant, autosomal recessive, and X-linked inheritance patterns. Recent studies have identified mutations in the ARL3 gene as a causative factor in both syndromic and non-syndromic forms of RP, including autosomal dominant and recessive cases. ARL3 encodes a small GTPase that plays a crucial role in intracellular trafficking, particularly within photoreceptors. This process is critical for maintaining ciliary function and phototransduction. Here, we investigate the pathogenic mechanisms of the ARL3 c.199G>C (p.Asp67His) variant identified in individuals from a four-generation family. We show that mutant ARL3 disrupts normal protein expression and affects ciliogenesis. Clinically affected individuals showed a non-syndromic retinal degenerative RP phenotype, with marked intrafamilial heterogeneity, ranging from extensive retinal atrophy to the absence of clinical manifestation, independent of age. This report highlights the incomplete penetrance and variable expressivity associated with the ARL3 variant and emphasizes the value of combining molecular diagnostics with functional validation to expedite molecular diagnosis.

A class of retinal dystrophies known as retinitis pigmentosa (RP) is caused by the loss of photoreceptor cells. RP can be genetically transmitted as an autosomal dominant, autosomal recessive, or X-linked trait. About one-third of genes implicated in retinal degeneration encode for proteins whose functional dysregulation affects the "connecting cilium" in photoreceptors, altering its structure and function. Here we report on a 33-year-old woman who was referred for clinical genetic testing following a previous diagnosis of degenerative retinopathy, which was not informative. She was enrolled in a research program dedicated to undiagnosed retinal disorders, where a whole genome sequencing approach was employed to understand the underlying genetic basis. The genomic analysis documented the occurrence of compound heterozygosity for two functionally relevant missense variants in BAIAP3, which encodes a protein with a well-documented role in SNARE-mediated trafficking and ciliogenesis. Confocal microscopy analysis showed elongated cilia in patient-derived and BAIAP3-depleted fibroblasts compared to control cells. Real-time PCR analyses showed a consistent significant reduction of GLI1 mRNA levels in patient-derived and BAIAP3-depleted cells, both in basal conditions and after treatment with Smoothened agonist, SAG, indicating Sonic hedgehog signaling dysregulation. Collectively, these data suggest that biallelic loss-of-function variants of BAIAP3 may cause photoreceptor degeneration and underlie isolated RP.

Background/Objectives: Virus-like particles (VLPs) represent an attractive platform for delivering vaccine formulations, combining a high biosafety profile with a potent immune-stimulatory ability. VLPs are non-infectious, non-replicating, self-assembling nanostructures that can be exploited to efficiently expose membrane-tethered glycoproteins such as the SARS-CoV-2 Spike (S) protein, the main target of approved preventive vaccines. Here, we describe the development and preclinical validation of Simian Immunodeficiency Virus (SIV)-based GFP-labeled VLPs displaying S from the B.1.617.2 (Delta) variant (VLP/S-Delta) for inducing persistent anti-SARS-CoV-2 neutralizing antibodies (nAbs) and S-specific T cell responses in mice. Methods: SIV-derived VLP/S-Delta were produced by co-transfecting a plasmid expressing SIVGag-GFP, required for VLP assembly and quantification by flow virometry, a plasmid encoding the Delta S protein deleted in the cytoplasmic tail (CT), to improve membrane binding, and a VSV.G-expressing plasmid, to enhance VLP uptake. Recovered VLPs were titrated by flow virometry and characterized in vitro by transmission electron microscopy (TEM) and confocal microscopy (CLSM). BALB/c mice were immunized intramuscularly with VLP/S-Delta following a prime–boost regimen, and humoral and cellular immune responses were assessed. Results: VLP/S-Delta were efficiently pseudotyped with CT-truncated S-Delta. After BALB/c priming, VLP/S-Delta elicited both specific anti-RBD IgGs and anti-Delta nAbs that significantly increased after the boost and were maintained over time. The prime–boost vaccination induced similar levels of cross-nAbs against the ancestral Wuhan-Hu-1 strain as well as cross-nAbs against Omicron BA.1, BA.2 and BA.4/5 VoCs, albeit at lower levels. Moreover, immunization with VLP/S-Delta induced S-specific IFNγ-producing T cells. Conclusions: These data suggest that SIV-based VLPs are an appropriate delivery system for the elicitation of efficient and sustained humoral and cellular immunity in mice, paving the way for further improvements in the immunogen design to enhance the quality and breadth of immune responses against different viral glycoproteins.

The CCL2/CC chemokine receptor 2 axis plays key roles in the pathogenesis of HIV-1 infection. We previously reported that exposure of monocyte-derived macrophages to CCL2 neutralizing antibody (αCCL2 Ab) restricted HIV-1 replication at postentry steps of the viral life cycle. This effect was associated with induction of transcripts coding for innate antiviral proteins, including APOBEC3A and RSAD2. This study aimed at identifying the signaling pathways involved in induction of these factors by CCL2 blocking in monocyte-derived macrophages. Through a combination of pharmacologic inhibition, quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal laser-scanning microscopy, we demonstrated that CCL2 neutralization activates the canonical NF-κB and JAK/STAT pathways, as assessed by time-dependent phosphorylation of IκB, STAT1, and STAT3 and p65 nuclear translocation. Furthermore, pharmacologic inhibition of IκB kinase and JAKs strongly reduced APOBEC3A and RSAD2 transcript accumulation elicited by αCCL2 Ab treatment. Interestingly, exposure of monocyte-derived macrophages to αCCL2 Ab resulted in induction of IL-6 family cytokines, and interference with glycoprotein 130, the common signal-transducing receptor subunit shared by these cytokines, inhibited APOBEC3A and RSAD2 upregulation triggered by CCL2 neutralization. These results provide novel insights into the signal transduction pathways underlying the activation of innate responses triggered by CCL2 neutralization in macrophages. Since this response was found to be associated with protective antiviral effects, the new findings may help design innovative therapeutic approaches targeting CCL2 to strengthen host innate immunity.

IntroductionExtracellular vesicles of Natural Killer cells (NKEV) exert an antitumor effect towards hematopoietic and solid tumors and have an immune modulating effect, suggesting a promising role in immune and biotherapy. In this study, a continuation of our former works, we demonstrated a network by mass spectrometry analysis between NKEV protein cargo and antitumor effects. Human healthy NKEV, both exosomes and microvesicles, have a significant and direct cytotoxic effect against human B cell lymphoma in in vitro and in vivo conditions.MethodsWe isolated extracellular vesicles from in vitro amplified healthy human NK cells and their treatment efficacy was monitored by cytometry analyses, in vivo MRI/MRS measurements, ex vivo MRS analyses and immunohistochemistry.ResultsWe observed a remarkable NKEV cytotoxic effect, mainly by apoptosis, on B cell lymphoma in vitro when exosomes and microvesicles were administered simultaneously. In vivo results showed metabolic alterations in SCID mice xenografts after NKEV treatment, associated with a significant reduction of tumor growth (64%). In the in vivo1H MR spectra we found a significant increase in the tumor lipid/lactate and in taurine signals, both considered as apotosis markers. Ex vivo lymphoma metabolomics revealed a significant increase in fatty acid (FA) pool and decrease in unsaturated and mono-unsaturated FA in treated groups, as compared to control one, thus suggesting an alteration of tumor homeostasis. Immunohistochemistry analyses confirmed the reduction of B-cell lymphoma proliferation rate, as well as the induction of apoptosis following the NKEV treatment. ConclusionsThis study underscore the importance of NKEV as a novel biological acellular tool for B-cell lymphoma treatment, probably having a greater effect on combined treatment regimens. These nanovesicles have an extraordinary potential in innovative cancer immunotherapy, representing a safe and efficient tool naturally circulating in healthy individuals and ready to maintain the immune homeostasis, and therefore a good organism healthy state.

The vacuolar H & thorn;-ATPase & thorn;-ATPase (V-ATPase) is a functionally conserved multimeric complex localized at the membranes of many organelles where its proton-pumping action is required for proper lumen acidification. The V-ATPase complex is composed of several subunits, some of which have been linked to human disease. We and others previously reported pathogenic dominantly acting variants in ATP6V1B2, the gene encoding the V1B2 subunit, as underlying a clinically variable phenotypic spectrum including dominant deafness-onychodystrophy (DDOD) syndrome, Zimmermann-Laband syndrome (ZLS), and deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures (DOORS) syndrome. Here, we report on an individual with features fitting DOORS syndrome caused by dysregulated ATP6V1C1 function, expand the clinical features associated with ATP6V1B2 pathogenic variants, and provide evidence that these ATP6V1C1/ATP6V1B2 amino acid substitutions result in a gain-of-function mechanism upregulating V-ATPase function that drives increased lysosomal acidification. We demonstrate a disruptive effect of these ATP6V1B2/ATP6V1C1 / ATP6V1C1 variants on lysosomal morphology, localization, and function, resulting in a defective autophagic flux and accumulation of lysosomal substrates. We also show that the upregulated V-ATPase function affects cilium biogenesis, further documenting pleiotropy. This work identifies ATP6V1C1 as new gene associated with a neurodevelopmental phenotype resembling DOORS syndrome, documents the occurrence of a phenotypic continuum between ZLS, and DDOD and DOORS syndromes, and classify these conditions as lysosomal disorders.

Background: Breast cancer represents one of the leading causes of death among women. Surgery can be effective, but once breast cancer has metastasized, it becomes extremely difficult to treat. Conventional therapies are associated with substantial toxicity and poor efficacy due to tumor heterogeneity, treatment resistance and disease relapse. Moreover, immune checkpoint blockade appears to offer limited benefit in breast cancer. The poor tumor immunogenicity and the immunosuppressive tumor microenvironment result in scarce T-cell infiltration, leading to a low response rate. Thus, there is considerable interest in the development of improved active immunotherapies capable of sensitizing a patient’s immune system against tumor cells. Methods: We evaluated the in vitro anti-tumor activity of a personalized vaccine based on dendritic cells generated in the presence of interferon (IFN)-α and granulocyte-macrophage colony-stimulating factor (IFN-DC) and loaded with an oxidized lysate from autologous tumor cells expanded as 3D organoid culture maintaining faithful tumor antigenic profiles. Results: Our findings demonstrate that stimulation of breast cancer patients’ lymphocytes with autologous IFN-DC led to efficient Th1-biased response and the generation in vitro of potent cytotoxic activity toward the patients’ own tumor cells. Conclusions: This approach can be potentially applied in association with checkpoint blockade and chemotherapy in the design of new combinatorial therapies for breast cancer.

Bardet–Biedl syndrome (BBS) is a rare recessive multisystem disorder characterized by retinitis pigmentosa, obesity, postaxial polydactyly, cognitive deficits, and genitourinary defects. BBS is clinically variable and genetically heterogeneous, with 26 genes identified to contribute to the disorder when mutated, the majority encoding proteins playing role in primary cilium biogenesis, intraflagellar transport, and ciliary trafficking. Here, we report on an 18-year-old boy with features including severe photophobia and central vision loss since childhood, hexadactyly of the right foot and a supernumerary nipple, which were suggestive of BBS. Genetic analyses using targeted resequencing and exome sequencing failed to provide a conclusive genetic diagnosis. Whole-genome sequencing (WGS) allowed us to identify compound heterozygosity for a missense variant and a large intragenic deletion encompassing exon 12 in BBS9 as underlying the condition. We assessed the functional impact of the identified variants and demonstrated that they impair BBS9 function, with significant consequences for primary cilium formation and morphology. Overall, this study further highlights the usefulness of WGS in the diagnostic workflow of rare diseases to reach a definitive diagnosis. This report also remarks on a requirement for functional validation analyses to more effectively classify variants that are identified in the frame of the diagnostic workflow.

Supplementary Figure 3 from Phosphatidylcholine-Specific Phospholipase C Activation in Epithelial Ovarian Cancer Cells

Supplementary Figure 2 from Phosphatidylcholine-Specific Phospholipase C Activation in Epithelial Ovarian Cancer Cells

Supplementary Figure 1 from Phosphatidylcholine-Specific Phospholipase C Activation in Epithelial Ovarian Cancer Cells

Kinesins are motor proteins involved in microtubule (MT)-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as 'ciliopathies.' Among kinesins, kinesin-1 is the most abundant MT motor for transport of cargoes towards the plus end of MTs. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.

Supplementary Figure Legends 1-3 from Phosphatidylcholine-Specific Phospholipase C Activation in Epithelial Ovarian Cancer Cells

Nanocellulose (NC) is an emerging material in the food sector with several prospective application areas. Three main types of NC exist, i.e. bacterial NC (BNC), nanofibrillated cellulose (NFC), and cellulose nanocrystals (CNC). The biological sources and processing conditions affect several physicochemical parameters of NC. In the present project, a NAM-based IATA for addressing data gaps in the assessment of potential hazards associated to NC oral exposure was considered. This IATA focused on three main pillars, i.e. (i) assessment of the uptake and potential crossing of the intestinal barrier by NC, (ii) assessment of local effects, including inflammation and genotoxicity, on the gastrointestinal epithelia, and (iii) assessment of any digestion or degradation of NC by the human microbiome. Eight NC samples belonging to the three NC types, plus a comparator in the micro-range, were selected as study materials and submitted to a thorough physicochemical characterisation. A battery of in vitro tests was used to provide insight into NC hazard and mode of action according to a tiered approach, which lead to selection of three materials belonging to the three main NC types for in depth-testing. Cell uptake of these materials was demonstrated, and such uptake was greater in a triculture model, which better simulates the barrier properties of the human intestinal epithelium, as compared to Caco-2 monolayers. Uptake was the greatest in repeated exposure conditions, in which intestinal barrier crossing was demonstrated for CNC. Pro-inflammatory responses accompanied by massive NC uptake in macrophages, indicative for potential immunotoxicological effects, and barrier function impairment were observed, whereas no indications for genotoxicity were obtained. Finally, no formation of smaller particles following colonic fermentation of NC was observed. For the integration of these results in regulatory hazard assessment of NC after oral exposure, prospective use of NC as novel food or as food additive was considered.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.