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.

Somatostatin receptor ligands (SRLs) with high affinity for somatostatin receptors 2 and 5 (SSTR2 and SSTR5) are poorly efficacious in NF-PitNETs, expressing high levels of SSTR3. ITF2984 is a pan-SSTR ligand with high affinity for SSTR3, able to induce SSTR3 activation and to exert antitumoral activity in the MENX rat model. The aim of this study was to test ITF2984’s antiproliferative and proapoptotic effects in NF-PitNET primary cultured cells derived from surgically removed human tumors and to characterize their SSTR expression profile. We treated cells derived from 23 NF-PitNETs with ITF2984, and a subset of them with octreotide, pasireotide (SRLs with high affinity for SSTR2 or 5, respectively), or cabergoline (DRD2 agonist) and we measured cell proliferation and apoptosis. SSTR3, SSTR2, and SSTR5 expression in tumor tissues was analyzed by qRT-PCR and Western blot. We demonstrated that ITF2984 reduced cell proliferation (−40.8 (17.08)%, p < 0.001 vs. basal, n = 19 NF-PitNETs) and increased cell apoptosis (+41.4 (22.1)%, p < 0.001 vs. basal, n = 17 NF-PitNETs) in all tumors tested, whereas the other drugs were only effective in some tumors. In our model, SSTR3 expression levels did not correlate with ITF2984 antiproliferative nor proapoptotic effects. In conclusion, our data support a possible use of ITF2984 in the pharmacological treatment of NF-PitNET.

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. The upregulation of the bombesin receptor 2 (BB2) in several malignancies and the advantages offered by peptide drug conjugates over antibody drug conjugates in terms of production and tumour targeting motivated us to synthesise and test bombesin conjugates armed with the tubulin binder monomethyl auristatin E. The widely used Val-Cit-PABC was initially included as cathepsin cleavable self-immolative linker for the release of the free drug. However, the poor stability of the Val-Cit-conjugates in mouse plasma encouraged us to consider the optimised alternatives Glu-Val-Cit-PABC and Glu-Gly-Cit-PABC. Conjugate BN-EVcM1, featuring Glu-Val-Cit-PABC, combined suitable stability (t(½) in mouse and human plasma: 8.4 h and 4.6 h, respectively), antiproliferative activity in vitro (IC50 = 29.6 nM on the human prostate cancer cell line PC-3) and the full release of the free payload within 24 h. Three conjugates, namely BN-EGcM1, BN-EVcM1 and BN-EVcM2, improved the accumulation of MMAE in PC-3 human prostate cancer xenograft mice models, compared to the administration of the free drug. Among them, BN-EVcM1 also stood out for the significantly extended survival of mice in in vivo acute efficacy studies and for the significant inhibition of the growth of a PC-3 tumour in mice in both acute and chronic efficacy studies.

Background: Somatostatin analogs (SSAs) binding to and activating somatostatin receptors (SSTRs) have been extensively used for the treatment of neuroendocrine tumors (NETs). The currently approved synthetic SSAs have high affinity for SSTR2 (octreotide/lanreotide) or for SSTR2 and SSTR5 (pasireotide). These agents have shown symptom control and antiproliferative effects in subsets of NET patients and this was associated with the expression of the targeted SSTRs. Pancreatic NETs (Pan-NETs) are uncommon tumors with a propensity to metastasize. For unresectable advanced Pan-NETs expressing SSTRs, SSAs are the first-line medical therapy. Pan-NETs express mainly SSTR1, SSTR2, and SSTR3 and thus should respond to agonists targeting SSTR3. Summary: We evaluated the efficacy of ITF2984, a novel multireceptor agonist with specificity for SSTR3, against PanNET cells representative of well-differentiated, functioning tumors, and expressing high levels of SSTR3. The effect of ITF2984 on cell proliferation/viability and on its ability to promote apoptosis and suppress hormone secretion was evaluated in 2D and 3D organotypic culture systems. Pasireotide was tested in parallel for comparative purposes. Key Message: We found that ITF2984 is as effective as pasireotide at inhibiting both proliferation/viability and hormone secretion, as well as at inducing apoptosis of PanNET cells grown as both 2D monolayers and 3D spheroids. High-dose ITF2984 elicits structural alterations in Pan-NET 3D spheroids compatible with cell death more effectively than pasireotide. Altogether, ITF2984 may represent a useful alternative to pasireotide for the medical treatment of PanNETs and other tumors with elevated SSTR3 expression.

Somatostatin receptor (SSTR) agonists have been extensively used for treating neuroendocrine tumors. Synthetic therapeutic agonists showing selectivity for SSTR2 (Octreotide) or for SSTR2 and SSTR5 (Pasireotide) have been approved for the treatment of patients with acromegaly and Cushing's syndrome, as their pituitary tumors highly express SSTR2 or SSTR2/SSTR5, respectively. Nonfunctioning pituitary adenomas (NFPAs), which express high levels of SSTR3 and show only modest response to currently available SSTR agonists, are often invasive and cannot be completely resected, and therefore easily recur. The aim of the present study was the evaluation of ITF2984, a somatostatin analog and full SSTR3 agonist, as a new potential treatment for NFPAs. ITF2984 shows a 10-fold improved affinity for SSTR3 compared to Octreotide or Pasireotide. Molecular modeling and NMR studies indicated that the higher affinity for SSTR3 correlates with a higher stability of a distorted β-I turn in the cyclic peptide backbone. ITF2984 induces receptor internalization and phosphorylation, and triggers G-protein signaling at pharmacologically relevant concentrations. Furthermore, ITF2984 displays antitumor activity that is dependent on SSTR3 expression levels in the MENX (homozygous mutant) NFPA rat model, which closely recapitulates human disease. Therefore, ITF2984 may represent a novel therapeutic option for patients affected by NFPA.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Histone deacetylase 6 (HDAC6) is a peculiar HDAC isoform whose expression and functional alterations have been correlated with a variety of pathologies such as auto immune disorders, neurodegenerative diseases, and cancer. It is primarily a cytoplasmic protein, and its deacetylase activity is focused mainly on nonhistone substrates such as tubulin, heat shock protein (HSP)90, Foxp3, and cortactin, to name a few. Selective inhibition of HDAC6 does not show cytotoxic effects in healthy cells, normally associated with the inhibition of Class I HDAC isoforms. Here, we describe the design and synthesis of a new class of potent and selective HDAC6 inhibitors that bear a pentaheterocyclic central core. These compounds show a remarkably low toxicity both in vitro and in vivo and are able to increase the function of regulatory T cells (Tregs) at well-tolerated concentrations, suggesting a potential clinical use for the treatment of degenerative, autoimmune diseases and for organ transplantation.

Tumor targeting has emerged as an advantageous approach to improving the efficacy and safety of cytotoxic agents or radiolabeled ligands that do not preferentially accumulate in the tumor tissue. The somatostatin receptors (SSTRs) belong to the G-protein-coupled receptor superfamily and they are overexpressed in many neuroendocrine tumors (NETs). SSTRs can be efficiently targeted with octreotide, a cyclic octapeptide that is derived from native somatostatin. The conjugation of cargoes to octreotide represents an attractive approach for effective tumor targeting. In this study, we conjugated octreotide to cryptophycin, which is a highly cytotoxic depsipeptide, through the protease cleavable Val-Cit dipeptide linker using two different self-immolative moieties. The biological activity was investigated in vitro and the self-immolative part largely influenced the stability of the conjugates. Replacement of cryptophycin by the infrared cyanine dye Cy5.5 was exploited to elucidate the tumor targeting properties of the conjugates in vitro and in vivo. The compound efficiently and selectively internalized in cells overexpressing SSTR2 and accumulated in xenografts for a prolonged time. Our results on the in vivo properties indicate that octreotide may serve as an efficient delivery vehicle for tumor targeting.

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1 beta and other cytokines at 25-100 nM; at concentrations >200 nM, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC50 of 285 nM). Here we compared the production of IL-1 beta, IL-1 alpha, TNF alpha, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nM; at 1000 nM, the secretion of IL-1 beta was reduced by 76%, secretion of TNF alpha was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1 alpha were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100-1000 nM. Gene expression of TNF alpha was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1 beta and TNF alpha was modestly reduced, IFN gamma production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1 beta independent of inhibition of caspase-1 activity; however, synthesis of the IL-1 beta precursor was reduced by 40% without significant decrease in IL-1 beta mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNF alpha by 85% and reduced IL-1 beta by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.

SummaryInduction of fetal haemoglobin (HbF) is a promising therapeutic approach for the treatment of β‐thalassaemia and sickle cell disease (SCD). Several pharmacological agents, such as hydroxycarbamide (HC) and butyrates, have been shown to induce the γ‐globin genes (HBG1, HBG2). However, their therapeutic use is limited due to weak efficacy and an inhibitory effect on erythroid differentiation. Thus, more effective agents are needed. The histone deacetylase (HDAC) inhibitors are potential therapeutic haemoglobin (Hb) inducers able to modulate gene expression through pleiotropic mechanisms. We investigated the effects of a HDAC inhibitor, Givinostat (GVS), on erythropoiesis and haemoglobin synthesis and compared it with sodium butyrate and HC. We used an in vitro erythropoiesis model derived from peripheral CD34+ cells of healthy volunteers and SCD donors. GVS effects on erythroid proliferation and differentiation and on Hb synthesis were investigated. We found that GVS at high concentrations delayed erythroid differentiation with no specific effect on HBG1/2 transcription. At a low concentration (1 nmol/l), GVS induced Hb production with no effects on cells proliferation and differentiation. The efficacy of GVS 1 mol/l in Hb induction in vitro was comparable to that of HC and butyrate. Our results support the evaluation of GVS as a new candidate molecule for the treatment of the haemoglobinophathies due to its positive effects on haemoglobin production at low and non‐toxic concentrations.

ITF2357 (givinostat) is a histone deacetylase inhibitor with antiinflammatory properties at low nanomolar concentrations. We report here a phase I safety and pharmacokinetics trial in healthy males administered 50, 100, 200, 400 or 600 mg orally. After 50mg, mean maximal plasma concentrations reached 104 nmol/L 2 h after dosing, with a half-life of 6.9 h. After 100 mg, maximal concentration reached 199 nmol/L at 2.1 h with a half-life of 6.0 h. Repeat doses for 7 consecutive days of 50, 100 or 200 mg resulted in nearly the same kinetics. There were no serious adverse effects (AEs) and no organ toxicities. However, there was a dose-dependent but transient fall in platelets. After 7 daily doses of 50 or 100 mg, the mean decrease in platelets of 17 and 25% was not statistically significant and returned to baseline within 14 d. Blood removed from the subjects after oral dosing was cultured ex vivo with endotoxin, and the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-1Ra, interferon (IFN)-γ and IL-10 was determined. Maximal reduction in IL-1β, TNFα, IL-6 and IFNγ was observed 4 h after dosing but returned to baseline at 12 h. There was no significant reduction in IL-1Ra or IL-10. With daily dosing, the fall in cytokine production in blood cultures observed on day 7 was nearly the same as that of the first day. We conclude that dosing of 50 or 100 mg ITF2357 is safe in healthy humans and transiently but repeatedly reduces the production of proinflammatory cytokines without affecting production of antiinflammatory cytokines.

A number of Lys-Pro-containing short peptides have been described as possessing a variety of biological activities in vitro. Because of limited metabolic stability, however, their efficacy in vivo is uncertain. To exploit the pharmacological potential of Lys-Pro-containing short peptides, we synthesized a series of chemically modified forms of these peptides. One of them, ITF1697 (Gly-(Nα-Et)Lys-Pro-Arg) was stable in vivo and particularly efficacious in experimental models of disseminated endotoxemia and of cardiovascular disorders. Using intravital fluorescence microscopy, we studied the peptide cellular and molecular basis of protection in the Syrian hamster cheek pouch microcirculation subjected to ischemia/reperfusion (I/R) and in pressure elevation-induced proinflammatory responses in isolated Sprague-Dawley rat lungs. Continuous intravenous infusion of ITF1697 at 0.1 to 100 µg/kg/min nearly completely protected the cheek pouch microcirculation from I/R injury as measured by decreased vascular permeability and increased capillary perfusion. Adhesion of leukocytes and platelets to blood vessels was strongly inhibited by the peptide. ITF1697 exerted its activity at the early stages of endothelial activation and inhibited P-selectin and von Willebrand factor secretion. Further mechanistic studies in the rat lung preparation revealed that the peptide inhibited the intracellular Ca 2+ -dependent fusion of Weibel-Palade bodies with the plasma membrane. The ability of ITF1697 to inhibit the early functions of activated endothelial cells, such as the exocytosis of Weibel-Palade bodies, represents a novel and promising pharmacological tool in model of pathologies of a variety of microvascular disorders.

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-α (TNFα) at 10 to 22 nM, the release of intracellular interleukin (IL-1α at 12 nM, the secretion of IL-1β at 12.5 to 25 nM, and the production of interferon-γ (IFNγ) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNγ and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFα. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFα and IFNγ mRNA but no effect on IL-1β or IL-8 levels. Real-time PCR confirmed the reduction in TNFα RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFα and IFNγ by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.