Novel Flavonoid Derivatives Show Potent Efficacy in Human Lymphoma Models

ABSTRACT

Background

Despite significant therapeutic progress, many lymphoma subtypes remain difficult to manage due to resistance, relapse, and dose‐limiting toxicity.

Methods

To elucidate the mechanism of action of the semi‐synthetic flavonoid derivative (SND) compounds, we conducted a screening of cancer cell lines using proliferation, cell cycle, and apoptosis assays. We then performed computational modeling of the compounds’ binding to tubulin, and finally evaluated in vivo activity using nanoNail technology alongside xenograft experiment.

Results

Here, we describe a series of SNDs that exhibit low‐nanomolar to picomolar cytotoxicity across multiple lymphoma models, including those resistant to BTK and PI3K inhibitors. Mechanistic studies show that these compounds trigger robust apoptosis via cytoskeletal disruption and mitochondrial dysfunction. Notably, SND207 also potently inhibits Protein Kinase N1, suggesting a synergistic link between kinase blockade and cytoskeletal interference. High‐throughput profiling places them near classical microtubule agents, although tubulin assays indicate more nuanced mechanisms than straightforward stabilization or depolymerization. In murine xenografts, SND207 significantly reduced tumor burden, and its combination with a BTK inhibitor demonstrates potential synergy. Furthermore, localized NanoNail delivery achieves high intratumoral drug concentrations at low doses, underscoring a favorable therapeutic index.

Conclusions

Overall, these findings highlight the translational promise of the SND series for future studies in the lymphoma field.

Clinical Trial Registration

The authors have confirmed clinical trial registration is not needed for this submission.

1. Introduction

Lymphomas are among the most common tumors in men and women for incidence and death [1]. Indeed, despite the big improvements of the last decades, still too many patients affected by lymphomas remain difficult to treat and cure due to resistance, relapse, and dose‐limiting toxicity [2]. Therefore, there is an unmet need for new therapies.

Plant‐derived agents represent a pivotal resource in oncology, displaying considerable efficacy against both solid and hematological tumors. Key examples include paclitaxel and vincristine, which continue to see widespread clinical use due to their proven antineoplastic properties [3].

Flavonoids are polyphenolic compounds produced by various plant species and are classified into six chemical groups: isoflavonoids, flavanones, flavanols, flavonols, flavones, and anthocyanidins [4]. Flavonoids regulate reactive oxygen species (ROS) by acting as antioxidants under normal conditions and as pro‐oxidants in cancer cells [5]. Additionally, their effects on the cytoskeleton are significant [6]. Phytochemicals such as phenolic acids, flavonoids, diterpenes, triterpenes, saponins, and alkaloids disrupt the cytoskeleton, impairing cell division, proliferation, and mobility, ultimately leading to apoptosis [7]. Wogonin, chrysin, and quercetin are plant‐derived polyphenols known to modulate multiple molecular targets and induce apoptosis in human tumor cells [8].

Flavopiridol, a flavone synthesized from the natural product rohitukine, acts as a kinase inhibitor of cyclin‐dependent kinases and other kinases [9]. Flavopiridol downregulates anti‐apoptotic proteins associated with resistance to fludarabine and rituximab. This was observed in a phase I study of flavopiridol, fludarabine, and rituximab (FFR) in patients with mantle‐cell lymphoma (MCL), indolent B‐cell non‐Hodgkin's lymphomas, and chronic lymphocytic leukemia (CLL) [10]. While the flavonoids have beneficial chemopreventive properties, and some of them exhibit moderate anti‐cancer activities in vitro, their clinical application is so far limited because of suboptimal bioavailability and lack of highly active molecules.

Protein Kinase N1 (PKN1), a serine/threonine kinase belonging to the protein kinase C (PKC)‐related kinase family, is involved in signal transduction, cytoskeletal organization, cell survival, and transcriptional regulation. Recent studies have demonstrated that PKN1 is overexpressed in multiple malignancies, contributing significantly to tumor progression by regulating cell proliferation, invasion, and metastasis [11, 12, 13].

Here, we describe a novel series of semi‐synthetic flavonoid derivatives (SNDs), which exhibit potent cytotoxicity across multiple B‐cell lymphoma subtypes—often at picomolar concentrations—and remain active in models resistant to BTK and PI3K inhibitors. Our mechanistic studies point to cytoskeletal disruption and mitochondrial dysfunction as central to their apoptotic effects, with some SND derivatives also strongly inhibiting PKN1. Our in vivo studies show that both a localized NanoNail delivery and systemic treatment yield significant tumor regression, and SND207 demonstrated minimal toxicity and enhanced efficacy when combined with ibrutinib. These findings collectively highlight the translational potential of the SND series to be further explored as novel therapeutic candidates for resistant forms of lymphoma.

2. Materials and Methods

2.1. Compounds

SND207, SND209, SND210, SND216, SND218, SND462, SND470, SND504, SND524, and SND562 were designed and provided by Floratek Pharma [14]. They were dissolved in DMSO as a 10 mM stock solution and then kept at ‐20°C.

2.2. Cell Lines and Culture Conditions

In total, 21 human lymphoma cell lines were used. Karpas422, Pfeiffer, SUDHL4, SUDHL6, Toledo, and WSU‐DLCL2 derived from germinal center B‐cell‐like (GCB) diffuse large B cell lymphoma (DLBCL). TMD8 and U2932 derived from activated B‐cell‐like (ABC) DLBCL. JEKO1, MINO, REC1, and SP53 are derived from mantle cell lymphoma (MCL). Karpas1718, SSK41, and VL51 derived from marginal zone lymphoma (MZL). MZL cell lines with acquired resistance to BTK and PI3K inhibitors (VL51 COP, VL51 IBR, VL51 IDE, and Karpas1718 IDE) were generated as previously described [15, 16, 17, 18].*

2.3. In Vitro Cytotoxic Activity

Cells were seeded in 96‐well plates and exposed to the compounds in a 1:10 dilution series ranging from 0.1 to 10000 nM. After 72 h, they were assayed by MTT [3‐(4,5‐dimethylthiazolyl‐2)‐2, 5‐diphenyltetrazoliumbromide] or counted using FACS. Sensitivity to drug treatments was evaluated by the IC₅₀, calculated based on the 4‐parameter logistic regression using the PharmacoGx R package [19].

2.4. Cell Cycle and Apoptosis Analysis

Cells were seeded at a 2 million per 10 mL density and subsequently treated with the compounds at the concentrations specified in Table S1. Cells were stained with Annexin V‐FITC (ThermoFisher Scientific, Waltham, MA, USA), washed, and then stained with propidium iodide (PI) for apoptosis assay. Cells were fixed with 70% cold ethanol before staining with PI and RNase (Sigma Aldrich, Buchs, Switzerland) treatment for the cell cycle. Acquisitions were carried out with a FACS Canto II instrument (BD Biosciences, Allschwil, Switzerland), and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR USA).

2.5. Correlations Assessment With Oncolines Profiler

The OncolinesProfiler (Oncolines B.V., Oss, The Netherlands) analysis was used to compare the IC₅₀ fingerprints of SND207, SND210, and SND562 to the IC50 fingerprints of pre‐profiled reference compounds (Table S2). All analyses were performed in the statistical software environment R [20].*

2.6. Molecular Docking

The molecular docking experiments were performed at Cambridge Crystallographic Data Centre (CCDC) (Cambridge, UK) using GOLD ensemble docking functionality [21]. The ligands’ structures were generated from SMILES using the CSD Conformer Generator [22]. The 10 most diverse RMSD conformers for each ligand were taken for the experiments. GoldScore [23] was used for the results ranking.*

2.7. Tubulin Polymerization Assay

Tubulin polymerization was assessed using a Tubulin Polymerization Assay Kit (Cytoskeleton Inc., #BK006P) at WuXi AppTec (Nantong, China). SND207, SND210, SND524, and SND562 were tested in a cell‐free assay according to the manufacturer's instructions. The absorbance was recorded in a kinetic mode at 340 nm for 90 min at 1 min intervals.

2.8. Mitochondrial‐ToxGlo Assay

Mitochondrial ToxGlo assay was used at WuXi AppTec (Nantong, China) to measure the cell membrane integrity and ATP levels in the SUDHL6 lymphoma cell line.*

2.9. Kinome Profiling

SND207 and SND562 were incubated with a panel of 408 kinases at 10 µM concentration and the ATP at Km for each kinase. The assay was performed by AssayQuant's KinSight Kinome Profiling service using the PhosphoSens format (Marlboro MA, USA). The inhibitory IC₅₀ of SND207 and SND562 on PKN1 was determined by 3‐fold titration, starting with 30 µM using the PhosphoSens platform.

2.10. NanoNail Technology

Athymic nude mice were subcutaneously inoculated with SUDHL6 cells (5 × 10⁶ cells/200 µL, Media/Matrigel 1:1). SND207, SND209, SND210, SND218, SND470 and SND562 were combined with high molecular weight polyethylene glycol (PEG) and loaded into the NanoNail (Kibur Medical, USA) at the concentrations specified in Table S5. When tumor volume reached 300–400 mm [3] one NanoNail containing 18 individual reservoirs with respective compounds was inserted in each tumor. Doxorubicin and paclitaxel were used as positive controls.*

2.11. Xenograft Model

All the procedures related to animal handling, care, and treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec (Nantong, China), following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).*

3. Results

3.1. In Vitro Anticancer Activity of SND Compounds

3.1.1. Flavonoid Derivatives Exhibit Potent In Vitro Anticancer Activity in Lymphoma Models

Seven SNDs, designed with the goal of enhancing cytotoxicity against cancer cells [24], displayed potent, dose‐dependent anti‐proliferative effects across 15 lymphoma cell lines, including models derived from GCB‐ and ABC‐DLBCL, MCL, and MZL.

Five of the SNDs exhibited low nanomolar activity (Figure 1 and Table S3). Among these, SND207 and SND210 emerged as the most potent, with median IC₅₀ values of 3.3 and 2.9 nM, respectively (Table S4). Notably, SND210 demonstrated remarkable efficacy against SUDHL4 (IC₅₀  =  97 pM) and SUDHL6 (IC₅₀  =  0.14 nM) cells (Figure 1 and Table S3).

FIGURE 1.

SND compounds exhibit strong in vitro anti‐lymphoma activity. Heatmap shows IC₅₀ values (log10, µM) from all seven compounds across 21 lymphoma cell lines. Including germinal center B‐cell like (GCB) diffuse large B cell lymphoma (DLBCL) (n = 6), activated B‐cell like (ABC) DLBCL (n = 2), mantle cell lymphoma (n = 4), marginal zone lymphoma (MZL) (n = 3) and MZL cell lines with acquired resistance to BTK (VL51 IBR) and PI3K (Karpas1718 IDE, VL51 IDE, VL51 COP) inhibitors (n = 4) and their parental (PAR) equivalents (n = 2).

Four additional cell lines resistant to BTK or PI3K inhibition (VL51 COP, VL51 IDE, VL51 IBR, and Karpas1718 IDE) were also included, along with their parental counterparts (VL51 PAR and Karpas1718 PAR), to evaluate efficacy in drug‐resistant settings. Both SND207 and SND210 maintained robust cytotoxic activity in cell lines resistant to ibrutinib (VL51 IBR, IC₅₀  =  7–8 nM), as well as to the PI3K inhibitors idelalisib (Karpas1718 IDE IC₅₀  =  1–2 nM; VL51 IDE IC₅₀  =  7–9 nM) and copanlisib (VL51 COP IC₅₀  =  17–26 nM) (Table S3). These observations underscore the potential of these derivatives to overcome common resistance pathways in clinical lymphoma treatment.

To understand the mechanism of action, cell cycle and apoptosis assays were performed. These assays revealed that all SND compounds exhibit cytotoxic properties. Specifically, SND207, SND210, SND462, and SND504 induced robust apoptosis in the sensitive SUDHL6 and WSU‐DLCL2 cell lines (Figure 2A,B and Figures S1 and S2) as well as in the resistant REC1 and VL51 models (Figures S3 and S4) at two‐times IC₅₀ doses (Table S1). Further examination in SUDHL6 cells showed that SND207, SND210, and SND562 elicit significant mitochondrial toxicity: these derivatives cause pronounced membrane rupture and ATP depletion, linking their cytotoxic mechanism to mitochondrial disruption (Figure 2C).

FIGURE 2.

SND compounds affect the cell cycle, and induce cell membrane rupture, apoptosis, and ATP depletion in SUDHL6 cells. Cell cycle changes (A) and percentage of apoptotic cells (B) in the SUDHL6 cell line after 72 h of treatment with SND207 (3 nM), SND462 (0.12 nM), SND210 (0.3 nM) and SND504 (0.05 nM). Cell membrane rupture and ATP content in SUDHL6 cells incubated with a concentration range of SND207, SND210, and SND562 for 4 h in the cell culture medium supplemented with galactose. Carbonyl cyanide 3‐chlorophenyl hydrazone (CCP) was used as a positive control (C).

3.1.2. SND462 and SND504 Exert Selective Activity in Specific Cell Models

Although SND462 and SND504 generally display lower overall potency than other SND derivatives (Table S3), they show remarkable selectivity and exert highly potent effects in particular cell lines. SUDHL6 appears especially vulnerable, exhibiting IC₅₀ values of 61 pM for SND462 and 22 pM for SND504, both in the picomolar range. WSU‐DLCL2 is also notably sensitive, although to a lesser degree. These exceptionally low IC₅₀ values highlight a potential subtype‐specific mechanism of action for SND462 and SND504, which appears to be most effective in the SUDHL6. Interestingly the cell lines harboring the EZH2‐Y641 mutation (SUDHL6, WSU‐DLCL2, and Karpas422) are overall more sensitive to SND462 and SND504 compared to EZH2 WT cell lines (Toledo and U2932).

3.2. Mechanism of Action of SND Compounds

3.2.1. Flavonoid Derivatives Target the Cytoskeleton

To explore potential mechanisms of action, the SND compounds were profiled using Oncolines Profiler, which compares IC₅₀ fingerprints against 213 pre‐characterized reference drugs. The analysis showed that SND derivatives cluster closely with known cytoskeleton‐targeting agents (e.g., vinblastine, vincristine, docetaxel, and paclitaxel) (Figure 3; Figures S5 and S6), strongly suggesting that they exert their anticancer effects by disrupting the cytoskeleton.

FIGURE 3.

SND207 cluster with cytoskeleton modulator drugs. Clustering tree of SND207 (marked with red arrow) based on the Oncolines activities of compounds profiled in a panel of cell lines. Each compound was assigned to one of 23 clusters and colored accordingly.

To validate this, molecular docking analyses were performed for SND218 and SND562 in the paclitaxel binding pocket of β‐tubulin. SND218 formed five hydrogen bonds via its trihydroxy‐4H‐1‐benzopyran moiety and is stabilized by hydrophobic interactions involving its aliphatic linker (Figure 4A,B). Similarly, SND562 established hydrogen bonds and additional hydrophobic contacts while also engaging in electrostatic interactions via a phosphorous atom (Figure 4C,D). Interestingly, SND218 and SND562 occupied distinct positions in the paclitaxel binding site (Figure 4A,C).

FIGURE 4.

Interaction of SND compounds with Tubulin. Molecular docking of SND218 (A, B) and SND562 (C, D) to paclitaxel binding site of b‐tubulin. Tubulin polymerization assay (E). Paclitaxel (1 µM) and vinblastine (3 µM) were used as positive controls. The peak max values of SND207, SND210, SND524, and SND526 were normalized to vehicle control. Statistics: two‐way ANOVA Compound vs. Vehicle (Paclitaxel and Vinblastine p < 0.001; SND524 at 0.06 µM and 0.02 µM p < 0.05).

Despite these strong in silico predictions, follow‐up tubulin polymerization assays using purified protein did not conclusively confirm direct microtubule stabilization or destabilization (Figure 4E). These results may indicate a more nuanced or indirect mechanism of cytoskeletal interference in living cells rather than straightforward tubulin polymerization blockade or enhancement. An effect of SND compounds on microtubule stability similar to that of paclitaxel cannot be excluded.

3.2.2. SND207 Is a Potent Inhibitor of PKN1

Additional mechanistic insights were gained by screening SND207 and SND562 against 408 kinases at a fixed compound concentration. SND207 demonstrated particularly strong selectivity for PKN1, inhibiting 87.6% of its activity with an IC₅₀ of approximately 140 nM (Figure S7B). These data suggest that PKN1 inhibition may play a central role in the cytotoxic mechanism of SND207 (Figure S7).

3.3. In Vivo Anticancer Activity of SND Compounds

3.3.1. Flavonoid Derivatives Delivered via NanoNail Technology Exhibit Potent In Vivo Anticancer Activity

We evaluated six compounds (SND207, SND209, SND210, SND218, SND470, and SND562) in a SUDHL6 xenograft model using Kibur Medical's NanoNail devices. Each device, containing 18 reservoirs, was inserted into the tumor via a biopsy needle. After 24 h, the surrounding tissue was collected, formalin‐fixed, and paraffin‐embedded for immunostaining with anti‐cleaved caspase‐3 (apoptosis) and anti‐Ki67 (proliferation).

Local drug diffusion (20–200 nM for SND207, SND209, SND470, and SND562; 40–160 nM for SND210 and SND218) enabled precise assessment of antiproliferative and proapoptotic effects in the tumor microenvironment. Notably, all SND derivatives achieved equal or superior efficacy at concentrations five‐fold lower than those used for paclitaxel or doxorubicin, underscoring their potent in vivo activity (Figure 5A,B and Table S5).

FIGURE 5.

SND derivatives cause a potent apoptosis activation and inhibition of cell proliferation in SUDHL6 xenografts. SND207, SND209, SND470, SND562 (0.2 µM), and SND210, SND218 (0.16 µM) increase the expression levels of apoptotic marker CC3 (A) and decrease the expression of cell proliferation marker ki67 (B). Dox—Doxorubicin (1 µM) and Pacli—Paclitaxel (1 µM) were used as positive controls.

3.3.2. In Vivo Anticancer Activity of SND207

Building on promising NanoNail results, SND207 was further examined in a CB17 SCID mouse model bearing SUDHL6 xenografts. The treatment with SND207 resulted in significant tumor growth inhibition (TGI) (Figure 6 and Table S6). Additionally, an exploratory combination study using ibrutinib (BTK inhibitor) with SND207 resulted in an even more pronounced TGI (Figure S8). All the treatments were well tolerated and did not cause significant weight loss (Figure S9).

FIGURE 6.

SND207 causes significant tumor growth inhibition in the SUDHL6 model in vivo. Tumor volume was measured over the course of treatment up to 21 days. Dosage: SND207 IP, QOD (0.3 mg/kg D0–15; 0.4 mg/kg D16–21). Statistics: two‐way ANOVA, Vehicle versus treatment group ****p‐value < 0.0001.

4. Discussion

The array of data presented throughout this study provides compelling evidence that these newly engineered flavonoid derivatives (SND series) represent a promising and versatile class of anti‐lymphoma agents. By integrating in vitro cytotoxicity assays, mechanistic evaluations (including cell cycle, apoptosis, mitochondrial toxicity, and kinase inhibition), and in vivo xenograft models, we tried to assemble a picture of how these compounds function and outperform traditional chemotherapy in certain settings.

Other attempts using azo‐flavonoid derivatives like 2‐aryl‐6,7‐methylenedioxyquinolin‐4‐one displayed selective cytotoxicity against solid cancer cell lines but at higher IC₅₀ values than the SND series described here. Mechanistic studies revealed that these new quinolone derivatives act as anti‐tubulin agents [25]. Additionally, 8‐substituted quercetin derivatives targeting β‐catenin/BCL9 inhibited colorectal cancer cell growth and suppressed Wnt signaling transactivation [26].

One of the most striking outcomes is the low‐nanomolar to picomolar cytotoxicity exhibited by certain SND compounds (notably SND207 and SND210) across multiple lymphoma subtypes, including difficult‐to‐treat variants such as ABC DLBCL and MCL. This potency is maintained or only modestly reduced in cell lines that have acquired resistance to approved kinase inhibitors (BTK or PI3K). Such broad‐spectrum effectiveness strongly suggests that these molecules target core oncogenic processes—such as cytoskeletal dynamics or survival signaling pathways—that remain essential in resistant cells. Two other compounds, SND462 and SND504, displayed a distinct activity profile; while showing moderate activity across most cell lines, they demonstrated exceptional potency in SUDHL6 cells. Their selective potency could be explained by the EZH2‐Y641 mutation, which occurs in 25% of GCB‐type DLBCL [27] and contributes to the proliferation of GCB‐DLBCL together with other oncogenic signals, such as translocations of the BCL2 oncogene and P53 loss, thus enhancing lymphoma cells survival [27, 28].

The selectivity of SND207 for PKN1 suggests promising mechanistic insights. PKN1 is a key regulator of the TRAF1‐NF‐κB signaling pathway, promoting cell survival and proliferation in cancers such as refractory CLL, where TRAF1 is frequently overexpressed [12, 29]. Additionally, PKN1 suppresses apoptosis by phosphorylating apoptosis‐related proteins, enabling cancer cells to evade normal regulatory mechanisms. Its involvement in the PI3K/AKT/mTOR and MAPK pathways, along with its regulation of RhoA‐mediated cytoskeletal dynamics, supports cell adhesion, migration, and potential metastasis [12, 13, 30]. While no direct PKN1 inhibitors are in clinical use, the phase III trial comparing R‐CHOP plus or minus enzastaurin, a potent PKC‐β and PI3K/AKT inhibitor tested in patients with relapsed or refractory DLBCL (NCT03263026), might provide relevant insights. Previous phase II studies showed enzastaurin's tolerability and prolonged failure‐free survival in relapsed/refractory DLBCL patients [31]. SND 207 is likely disrupting the PKN1‐TRAF1 axis, reducing pro‐survival proteins, by inducing apoptosis, and impairing cytoskeletal functions critical for migration and adhesion in lymphoma. Further studies are needed to elucidate its effects on lymphoma cell survival, proliferation, and migration.

An in vivo experiment, taking advantage of the NanoNail device, showed that the local release of SND207, SND209, SND210, SND218, SND470, and SND562 led to equal or superior anti‐tumor activity at concentrations five‐fold lower than what used for paclitaxel or doxorubicin.

Finally, systemic administration of SND207 achieved similarly notable tumor growth inhibition in SUDHL6 tumor‐bearing mice. An exploratory combination study with BTK inhibitor suggests that SND207 might have additive or synergistic effects when combined with other agents. This can be further explored in different lymphoma models that are particularly dependent on B‐cell receptor signaling or exhibit dysregulation in pathways involving BTK and cytoskeletal dynamics.

Conventional drugs such as paclitaxel, vincristine, and bortezomib cause neuropathy in about two‐thirds of patients within the first month of treatment. Recent studies suggest that this neuropathy is linked to signal transduction molecules, including protein kinase C and mitogen‐activated protein kinases [32]. Despite potent antitumor activity, the body weight post‐treatment with SND series compounds remained stable or near‐baseline, possibly indicating minimal systemic toxicity. Whether these SND derivatives truly offer reduced neuropathic risk requires dedicated follow‐up experiments, yet these preliminary tolerability data are promising.

Collectively, our findings establish this new series of SNDs as powerful and versatile anti‐lymphoma agents capable of simultaneous cytoskeletal disruption and selective kinase inhibition. By exerting low‐nanomolar to picomolar cytotoxicity across a range of lymphoma models, especially those resistant to BTK or PI3K blockade, SNDs could fill a critical gap in treating relapsed/refractory diseases. Their multi‐faceted mode of action, spanning tubulin interference, mitochondrial toxicity, and signaling inhibition, may enable them to bypass or overcome the compensatory pathways that often underlie therapeutic failure. The promising in vivo performance highlights the clinical potential of combining these SND molecules with existing targeted agents. Looking ahead, in‐depth toxicity assessments, particularly regarding neuropathy and extended pharmacokinetic evaluations, will be paramount for translating these compounds into clinical practice. Should these ongoing studies confirm efficacy and tolerability, the SND series could be a valuable addition to the lymphoma treatment armamentarium and extend its reach into other malignancies reliant on microtubule integrity and kinase‐driven survival pathways.

Author Contributions

Alberto J. Arribas, Eleonora Cannas, Guido J. R. Zaman, and Eugenio Gaudio performed experiments; Alberto J. Arribas, Eleonora Cannas, Paulina Biniecka, Eugenio Gaudio, Francesco Bertoni, Guido J. R. Zaman, Derya Unutmaz, and DFS analyzed data; Dan F. Stoicescu, Eugenio Gaudio, Alberto J. Arribas, and Francesco Bertoni contributed to the conception or design of the studies; Eugenio Gaudio, Paulina Biniecka, Francesco Bertoni, Derya Unutmaz, and Dan F. Stoicescu wrote the manuscript.

Conflicts of Interest

Eugenio Gaudio, Paulina Biniecka, and Dan F. Stoicescu are employees of Floratek Pharma SA. Eleonora Cannas, Guido J. R. Zaman, and Derya Unutmaz have declared no conflicts of interest. Alberto J. Arribas has received a travel grant from AstraZeneca and Floratek Pharma and serves as a consultant for PentixaPharm. Francesco Bertoni has received institutional research funds from ADC Therapeutics, Bayer AG, BeiGene, Floratek Pharma, Helsinn, HTG Molecular Diagnostics, Ideogen AG, Idorsia Pharmaceuticals Ltd., Immagene, ImmunoGen, Menarini Ricerche, Nordic Nanovector ASA, and Oncternal Therapeutics, Spexis AG. He has also received consultancy fees from BIMINI Biotech, Floratek Pharma, Helsinn, Immagene, Menarini, and Vrise Therapeutics. Additionally, he has received advisory board fees (paid to his institution) from Novartis, provided expert statements to HTG Molecular Diagnostics, and received travel grants from Amgen, AstraZeneca, and iOnctura.

Supporting information

Supporting File 1: jha270081‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article.