Search

Matteo Petrini,1,2 Wouter JM Lokerse,1 Agnieszka Mach,1 Martin Hossann,3 Olivia M Merkel,2 Lars H Lindner1 1Department of Internal Medicine III, University Hospital, Ludwig Maximilian University, Munich, Germany; 2Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig Maximilian University, Munich, Germany; 3Thermosome GmbH, Planegg, GermanyCorrespondence: Lars H LindnerLudwig Maximilian University, Department of Internal Medicine III, Klinikum Grosshadern, Marchioninistr. 15, Munich, 81377, GermanyTel +49-89-7095-4768Fax +49-89-7095-4776Email lars.lindner@med.uni-muenchen.dePurpose: Previous studies demonstrated the possibility of targeting tumor-angiogenic endothelial cells with positively charged nanocarriers, such as cationic liposomes. We investigated the active targeting potential of positively charged nanoparticles in combination with the heat-induced drug release function of thermosensitive liposomes (TSL). This novel dual-targeted approach via cationic TSL (CTSL) was thoroughly explored using either a novel synthetic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) or a conventional polyethylene glycol (PEG) surface modification. Anionic particles containing either DPPG2 or PEG were also included in the study to highlight difference in tumor enrichment driven by surface charge. With this study, we aim to provide a deep insight into the main differences between DPPG2- and PEG-functionalized liposomes, focusing on the delivery of a well-known cytotoxic drug (doxorubicin; DOX) in combination with local hyperthermia (HT, 41– 43°C).Materials and Methods: DPPG2- and PEG-based cationic TSLs (PG2-CTSL/PEG-CTSL) were thoroughly analyzed for size, surface charge, and heat-triggered DOX release. Cancer cell targeting and DOX delivery was evaluated by FACS, fluorescence imaging, and HPLC. In vivo particle behavior was analyzed by assessing DOX biodistribution with local HT application in tumor-bearing animals.Results: The absence of PEG in PG2-CTSL promoted more efficient liposome–cell interactions, resulting in a higher DOX delivery and cancer cell toxicity compared with PEG-CTSL. By exploiting the dual-targeting function of CTSLs, we were able to selectively trigger DOX release in the intracellular compartment by HT. When tested in vivo, local HT promoted an increase in intratumoral DOX levels for all (C)TSLs tested, with DOX enrichment factors ranging from 3 to 14-fold depending on the type of formulation.Conclusion: Cationic particles showed lower hemocompatibility than their anionic counterparts, which was partially mitigated when PEG was grafted on the liposome surface. DPPG2-based anionic TSL showed optimal local drug delivery compared to all other formulations tested, demonstrating the potential advantages of using DPPG2 lipid in designing liposomes for tumor-targeted applications.Keywords: thermosensitive liposomes, mild hyperthermia, cationic liposomes, dual tumor targeting, drug delivery, liposome functionalization

Drug product development of therapeutic antibody formulations is still dictated by the risk of protein particle formation during processing or storage, which can lead to loss of potency and potential immunogenic reactions. Since structural perturbations are the main driver for irreversible protein aggregation, the conformational integrity of antibodies should be closely monitored. The present study evaluated the applicability of a plate reader-based high throughput method for Intrinsic Tryptophan Fluorescence Emission (ITFE) spectroscopy to detect protein aggregation due to protein unfolding in high-concentrated therapeutic antibody samples. The impact of fluorophore concentration on the ITFE signal in microplate readers was investigated by analysis of dilution series of two therapeutic antibodies and pure tryptophan. At low antibody concentrations (< 5 mg/mL, equivalent to 0.8 mM tryptophan), the low inner filter effect suggests a quasi-linear relationship between antibody concentration and ITFE intensity. In contrast, the constant ITFE intensity at high protein concentrations (> 40 mg/mL, equivalent to 6.1 mM tryptophan) indicate that ITFE spectroscopy measurements of IgG1 antibodies are feasible in therapeutically relevant concentrations (up to 223 mg/mL). Furthermore, the capability of the method to detect low levels of unfolding (around 1 %) was confirmed by limit of detection (LOD) determination with temperature-stressed antibody samples as degradation standards. Change of fluorescence intensity at the maximum (ΔIaM) was identified as sensitive descriptor for protein degradation, providing the lowest LOD values. The results demonstrate that ITFE spectroscopy performed in a microplate reader is a valuable tool for high-throughput monitoring of protein degradation in therapeutic antibody formulations., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Prostate cancer develops through malignant transformation of the prostate epithelium in a stepwise, mutation-driven process. Although activator protein-1 transcription factors such as JUN have been implicated as potential oncogenic drivers, the molecular programs contributing to prostate cancer progression are not fully understood., Methods: We analyzed JUN expression in clinical prostate cancer samples across different stages and investigated its functional role in a Pten-deficient mouse model. We performed histopathological examinations, transcriptomic analyses and explored the senescence-associated secretory phenotype in the tumor microenvironment., Results: Elevated JUN levels characterized early-stage prostate cancer and predicted improved survival in human and murine samples. Immune-phenotyping of Pten-deficient prostates revealed high accumulation of tumor-infiltrating leukocytes, particularly innate immune cells, neutrophils and macrophages as well as high levels of STAT3 activation and IL-1β production. Jun depletion in a Pten-deficient background prevented immune cell attraction which was accompanied by significant reduction of active STAT3 and IL-1β and accelerated prostate tumor growth. Comparative transcriptome profiling of prostate epithelial cells revealed a senescence-associated gene signature, upregulation of pro-inflammatory processes involved in immune cell attraction and of chemokines such as IL-1β, TNF-α, CCL3 and CCL8 in Pten-deficient prostates. Strikingly, JUN depletion reversed both the senescence-associated secretory phenotype and senescence-associated immune cell infiltration but had no impact on cell cycle arrest. As a result, JUN depletion in Pten-deficient prostates interfered with the senescence-associated immune clearance and accelerated tumor growth., Conclusions: Our results suggest that JUN acts as tumor-suppressor and decelerates the progression of prostate cancer by transcriptional regulation of senescence- and inflammation-associated genes. This study opens avenues for novel treatment strategies that could impede disease progression and improve patient outcomes., (© 2024. The Author(s).)

Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse., (© 2024. The Author(s).)

Anaplastic large cell lymphoma (ALCL) is an aggressive, CD30 + T cell lymphoma of children and adults. ALK fusion transcripts or mutations in the JAK-STAT pathway are observed in most ALCL tumors, but the mechanisms underlying tumorigenesis are not fully understood. Here, we show that dysregulated STAT3 in ALCL cooccupies enhancers with master transcription factors BATF3, IRF4, and IKZF1 to form a core regulatory circuit that establishes and maintains the malignant cell state in ALCL. Critical downstream targets of this network in ALCL cells include the protooncogene MYC, which requires active STAT3 to facilitate high levels of MYC transcription. The core autoregulatory transcriptional circuitry activity is reinforced by MYC binding to the enhancer regions associated with STAT3 and each of the core regulatory transcription factors. Thus, activation of STAT3 provides the crucial link between aberrant tyrosine kinase signaling and the core transcriptional machinery that drives tumorigenesis and creates therapeutic vulnerabilities in ALCL., Competing Interests: Declaration of interests K.S. is a member of the scientific advisory board and has stock options in Auron Therapeutics and received grant funding from Novartis and KronosBio on topics unrelated to this paper. B.J.A. is a shareholder in Syros Pharmaceuticals. N.V.D. is currently an employee of Genentech, and is a stockholder in Roche. A.T.L. is a shareholder in LightHorse Therapeutics and is a consultant/advisory board member for LightHorse Therapeutics and Omega Therapeutics. M.W.Z. is currently an employee and shareholder in Foghorn Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Anaplastic large-cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by the oncogenic anaplastic lymphoma kinase (ALK), accounting for approximately 15% of all paediatric non-Hodgkin lymphoma. Patients with central nervous system (CNS) relapse are particularly difficult to treat with a 3-year overall survival of 49% and a median survival of 23.5 months. The second-generation ALK inhibitor brigatinib shows superior penetration of the blood-brain barrier unlike the first-generation drug crizotinib and has shown promising results in ALK+ non-small-cell lung cancer. However, the benefits of brigatinib in treating aggressive paediatric ALK+ ALCL are largely unknown. We established a patient-derived xenograft (PDX) resource from ALK+ ALCL patients at or before CNS relapse serving as models to facilitate the development of future therapies. We show in vivo that brigatinib is effective in inducing the remission of PDX models of crizotinib-resistant (ALK C1156Y, TP53 loss) ALCL and furthermore that it is superior to crizotinib as a second-line approach to the treatment of a standard chemotherapy relapsed/refractory ALCL PDX pointing to brigatinib as a future therapeutic option., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eμ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss., (© 2022 by The American Society of Hematology.)

Over the past 10 years, the interest in intranasal drug delivery in pharmaceutical R&D has increased. This review article summarises information on intranasal administration for local and systemic delivery, as well as for CNS indications. Nasal delivery offers many advantages over standard systemic delivery systems, such as its non-invasive character, a fast onset of action and in many cases reduced side effects due to a more targeted delivery. There are still formulation limitations and toxicological aspects to be optimised. Intranasal drug delivery in the field of drug development is an interesting delivery route for the treatment of neurological disorders. Systemic approaches often fail to efficiently supply the CNS with drugs. This review paper describes the anatomical, histological and physiological basis and summarises currently approved drugs for administration via intranasal delivery. Further, the review focuses on toxicological considerations of intranasally applied compounds and discusses formulation aspects that need to be considered for drug development., (© 2021. The Author(s).)

Personalized medicine aims to match the right drug with the right patient by using specific features of the individual patient's tumor. However, current strategies of personalized therapy matching provide treatment opportunities for less than 10% of patients with cancer. A promising method may be drug profiling of patient biopsy specimens with single-cell resolution to directly quantify drug effects. We prospectively tested an image-based single-cell functional precision medicine (scFPM) approach to guide treatments in 143 patients with advanced aggressive hematologic cancers. Fifty-six patients (39%) were treated according to scFPM results. At a median follow-up of 23.9 months, 30 patients (54%) demonstrated a clinical benefit of more than 1.3-fold enhanced progression-free survival compared with their previous therapy. Twelve patients (40% of responders) experienced exceptional responses lasting three times longer than expected for their respective disease. We conclude that therapy matching by scFPM is clinically feasible and effective in advanced aggressive hematologic cancers. SIGNIFICANCE: This is the first precision medicine trial using a functional assay to instruct n-of-one therapies in oncology. It illustrates that for patients lacking standard therapies, high-content assay-based scFPM can have a significant value in clinical therapy guidance based on functional dependencies of each patient's cancer. See related commentary by Letai, p. 290 . This article is highlighted in the In This Issue feature, p. 275 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

Anaplastic large-cell lymphoma (ALCL) is a T-cell malignancy driven in many cases by the product of a chromosomal translocation, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK activates a plethora of pathways that drive the hallmarks of cancer, largely signalling pathways normally associated with cytokine and/or T-cell receptor-induced signalling. However, NPM-ALK is also located in the nucleus and its functions in this cellular compartment for the most part remain to be determined. We show that ALCL cell lines and primary patient tumours express the transcriptional activator BRG1 in a NPM-ALK-dependent manner. NPM-ALK regulates expression of BRG1 by post-translational mechanisms dependent on its kinase activity, protecting it from proteasomal degradation. Furthermore, we show that BRG1 drives a transcriptional programme associated with cell cycle progression. In turn, inhibition of BRG1 expression with specific shRNA decreases cell viability, suggesting that it may represent a key therapeutic target for the treatment of ALCL.