The introduction of proteasome inhibitors (PIs) into multiple myeloma (MM) treatment has substantially improved therapeutic options and survival rates. However, the development of resistance to PIs as well as serious side effects of PI treatment continue to justify the search for effective, less burdensome combination therapies. Thus, we investigated the therapeutic potential of VS-4718 (dual pPYK2- and pFAK-inhibitor) alone and in combination with the PI carfilzomib (carf) in parental human MM cell lines (pHMCLs) and PI triple-resistant HMCLs (rHMCLs). VS-4718 reduced the viability in a concentration dependent manner in all pHMCLs and the response was independent of PYK2 expression or activation. A more than additive impact of the combination therapy on survival, measured by annexin V-FITC/PI staining, was observed in 5/7 pHMCLs. Titration experiments showed that VS-4718 in combination with low and sublethal doses of carf had a specific anti-tumor effect on pHMCLs but hardly affected peripheral blood mononuclear cells. In rHMCLs, addition of VS-4718 to carf re-sensitized cells to carf and revealed a more than additive reduction in cell survival. These findings suggest that VS-4718 together with low doses of carf could be an effective and low-toxic combination in MM, including PI-resistant, relapsed/refractory MM.

11516 Background: Patients with advanced or recurrent synovial sarcoma (SS) have limited treatment options that can provide sustained disease control beyond 6 mos. SS expresses multiple cancer testis antigens, including PRAME, which is expressed in >50 cancers. Anzutresgene autoleucel (anzu-cel, IMA203) is a PRAME-directed TCR T-cell therapy engineered to recognize an intracellular PRAME-derived peptide presented by HLA and initiate a potent and specific antitumor response. IMA203CD8 is a TCR T-cell therapy that additionally engineers a CD8 co-receptor to allow both CD4+ and CD8+ T cells to detect and destroy PRAME-expressing tumor cells that evade conventional therapeutic approaches. Here we present phase 1 results for the subset of pts with SS from IMA203-101 treated with anzu-cel or IMA203CD8 (hereafter TCR T). Methods: Pts were ≥18 y, HLA-A*02:01+, PRAME+, with R/R solid tumors, measurable disease (RECIST 1.1), ECOG PS 0-1, and no remaining SOC treatment options. Following leukapheresis and TCR T manufacture, pts underwent lymphodepletion (LD) with Cy (500 mg/m 2 × 4 d) and Flu (30 mg/m 2 × 4 d), followed by TCR T infusion +/- low-dose SUBQ IL-2. Results: As of Oct 27, 2025, 21 pts with SS were treated with anzu-cel (n=9) or IMA203CD8 (n=12). The most common TEAEs were LD-related cytopenias (100%). Cytokine release syndrome occurred in 18 pts (86%; G1: 38%, G2: 38%, G3: 10%). One event of G1 immune effector cell–associated neurotoxicity occurred (5%). Twenty pts were evaluable for efficacy and received a low median dose of 1.51 x 10 9 total TCR T cells (range: 0.35-9.36 x 10 9 ). These pts had a median sum of target lesions at baseline of 10.22 cm (range: 1.24-41.1 cm) and received a median of 2.5 prior lines of therapy (range: 1-5). At baseline, 65% had ≥2 previous lines of systemic therapy and 75% received prior radiotherapy. The confirmed objective response rate (cORR), including all pts evaluable for efficacy across escalating doses, was 50% (10/20), including 1 confirmed CR (8%) with IMA203CD8 at a dose of 2.05 x 10 9 total TCR T cells. Median tumor reduction for pts with confirmed objective response was -63.4%. Median duration of response at these escalating dose levels was 12.7 mo (mFU: 23 mo) with 5 responses lasting for ≥12 mo including 2 ongoing PRs at 27 and 23 mo. Median overall survival was 18 mo. Results include 1 pt who received 3 sequential TCR T-cell therapies: the MAGE-A4-targeted afamitresgene-autoleucel (BOR: SD; PFS: 21.4 mos), PRAME-targeted anzu-cel (BOR: cPR; PFS: 24.4 mo), and IMA203CD8 (BOR: cPR; PFS ongoing at 24.4 mo). Conclusions: PRAME-directed TCR T-cell therapies exhibited a safety profile consistent with previous observations, and despite low doses, promising antitumor activity with durable responses that deepened over time in pts with SS. These results warrant further investigation. Clinical trial information: NCT03686124 .

IMA401 is a T cell receptor (TCR)-based next-generation bispecific T cell engaging receptor (TCER) targeting an HLA-A*02:01-presented peptide derived from MAGE-A4/MAGE-A8 with its high-affinity TCR-based domain, incorporating a low-affinity T-cell-recruiting domain and an optimized Fc domain to prolong half-life. In this prespecified interim analysis of a phase 1 first-in-human trial, 61 patients with advanced solid tumors received intravenous IMA401 (0.0066 mg-2.5 mg) with or without pembrolizumab. The primary endpoint was determination of the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) of IMA401 monotherapy and in combination with pembrolizumab. Secondary objectives included safety and tolerability, antitumor activity and pharmacokinetics. The MTD was not reached as defined by the clinical trial protocol, and the RP2D was 1-2 mg IMA401 biweekly. Treatment-related adverse events (TRAEs) were well manageable; the most common any-grade TRAEs were cytokine release syndrome (38%, grades 1-2 only), transient lymphopenia (33%) and reversible neutropenia (31%). Five patients experienced dose-limiting toxicity (DLT) events primarily related to neutropenia. No further DLTs occurred in the RP2D range with dexamethasone premedication. One possibly-related death (pneumonia in a patient with rapidly progressing lung metastases) was reported outside RP2D at 2.5 mg IMA401. In the overall efficacy-evaluable population across all dose levels (n = 56), including low starting doses (from 0.0066 mg), the confirmed objective response rate (ORR) was 14% (8/56). In patients receiving IMA401 at the RP2D, an ORR of 20% (8/41) was observed across 15 different indications (post hoc analysis). In the largest subgroup of patients treated at RP2D, namely head and neck cancer, the ORR was 29% (4/14) with a median duration of response of 8.8 months. These findings show that the bispecific TCER platform has a manageable safety profile with mostly transient adverse events and promising antitumor activity at the RP2D of IMA401 with or without pembrolizumab. ClinicalTrials.gov identifier: NCT05359445 .

9508 Background: Pts with advanced melanoma frequently experience progressive disease (PD) with limited long-term survival following available therapies; there is a need for novel systemic treatments that can induce deep and durable responses. PRAME is a cancer-associated antigen expressed in >50 cancers, including melanoma. Anzu-cel (IMA203) is an autologous PRAME-directed TCR T-cell therapy engineered to recognize intracellular PRAME-derived peptides presented by HLA-A*02:01 and mediates a potent and specific anti-tumor immune response (Wermke et al. ASCO, 2025). A registration-directed phase 3 trial (SUPRAME; NCT06743126) is underway. Methods: IMA203-101 is an ongoing, multicenter, phase 1/2 trial evaluating anzu-cel in pts with advanced PRAME-positive solid tumors. Pts underwent lymphodepleting (LD) chemotherapy followed by a single infusion of anzu-cel +/- low-dose SUBQ IL-2. This analysis focuses on melanoma pts in phase 1b treated at RP2D. Data cutoff was Sep 24, 2025. Exploratory analyses evaluated lesion-level patterns of progression among pts evaluable per RECIST1.1. Results: As of data cutoff, 33 pts with melanoma were evaluable, incl. cutaneous (n=14), uveal (n=16), and other subtypes (n=3). Most frequent AEs were LD-related cytopenias. AEs of special interest included cytokine release syndrome (CRS), immune effector cell–associated neurotoxicity syndrome (ICANS) and hemophagocytic lymphohistiocytosis (HLH). CRS was predominantly low grade (G1: 36%, G2: 46%, G3: 18%) and occurred early after infusion, with incidence and severity decreasing over time. ICANS (G1: 6%, G3: 6%) and HLH (G2: 3%, G3: 3%) were infrequent. Confirmed ORR was 56% (cCR=1, cPR=17) and DCR was 91%. mDOR was 14.6 mos (4.2-38.2+), mPFS was 6.1 mos (1.4-39.6+), and mOS was 16.2 mos (2.4-39.6+). Responses were observed in target and nontarget lesions, with most pts demonstrating initial lesion responses within the first 6 weeks of treatment; median time to BOR of cPR/cCR was 1.4 mo (1.2-2.8). 25 pts were evaluable for patterns of PD. At the time of PD, 68% (17/25) developed new lesions (NL); 1 pt developed new central nervous system (CNS) lesions. PD via growth of existing lesions was observed in 56% (14/25); 24% (6/25) experienced PD by both growth of existing lesions and NLs. No specific pattern of PD was identifiable in those who achieved cPR compared to those who did not. Conclusions: Anzu-cel demonstrated favorable tolerability and induced clinically meaningful and durable responses. Exploratory analyses suggest that the CNS was not a common site for relapse. Future analyses will characterize response dynamics and progression patterns to better define scenarios in which progression following cPR remains clinically manageable, potentially supporting individualized decision-making regarding timing and necessity of subsequent systemic therapy. Clinical trial information: NCT06743126 .

2507 Background: IMA401 is a next-gen bispecific TCR-based T-cell engager designed to redirect T cells to antigen-positive cancer cells. It combines a high-affinity, highly specific TCR domain against an HLA-A*02:01-presented target peptide common to cancer-specific antigens MAGEA4 and MAGEA8 with a low-affinity T-cell–recruiting domain for improved tolerability and biodistribution, and an Fc part for half-life extension. IMA401-101 (NCT05359445) is a first-in-human phase 1a/b basket study evaluating IMA401 in pts with advanced solid tumors. Methods: Pts were ≥18 y, HLA-A*02:01+, MAGEA4+ and/or MAGEA8+, have R/R solid tumors, measurable disease (RECIST 1.1), ECOG performance status 0-2, and have exhausted SOC options. Dose escalation involved cohorts of 1-6 pts using adaptive design (BLRM). Doses ranged from 0.0066 mg - 2.5 mg IMA401 q2w (± pembrolizumab [pembro] q6w with 1.0 mg or 1.5 mg IMA401). Weekly step dosing of IMA401 was implemented for the first 2-3 doses at ≥1 mg . Primary endpoint was MTD assessed by BLRM and/or RP2D as monotherapy and in combination with pembro. Secondary endpoints included safety and initial antitumor activity (confirmed objective response rate [cORR] and disease control rate [DCR]). Results: As of Sep 26, 2025, 55 heavily pretreated pts across > 15 different tumor types with a median of 4 prior lines of therapy (range 1-9) received IMA401 ± pembro. Most frequent TRAEs were low-grade CRS (G1: 24%; G2: 11%; no ≥G3 events), expected and transient lymphopenia (any grade: 29%; ≥G3: 24% which typically improved within 1-3 days), and reversible neutropenia (any grade: 29%; ≥G3: 18%). No ICANS was observed. Tolerability of IMA401 + pembro (n = 9) was consistent with the IMA401 monotherapy safety profile. MTD was not reached (3 DLTs at 2.5 mg IMA401). RP2D range was determined to be 1-2 mg IMA401. Efficacy was evaluable in 38 pts receiving a target dose of ≥1 mg IMA401. Promising clinical activity was noted in pts with head and neck (HN) cancer (cORR: 25% [2/8]; DCR: 63% [5/8]) and melanoma (cORR: 29% [2/7]; DCR: 57% [4/7]) with duration of all confirmed responses beyond 6 months postinfusion. In squamous non-small cell lung cancer (sqNSCLC [n = 3]), 1 PR with reduction in all target lesions, 1 SD with OS of ~16 months and 1 PD with reduction in all liver target lesions were observed. After data cutoff, 2 more cPRs were observed in HN cancer resulting in a cORR of 33% (4/12). An updated full dataset will be presented. Conclusions: IMA401 demonstrated overall favorable tolerability without reaching formal MTD and encouraging antitumor activity in pts with HN cancer, melanoma, and sqNSCLC at RP2D range. Based on the promising results and preclinical proof of concept data, further development steps are being evaluated including a potentially synergistic combination with the PRAME-directed bispecific IMA402 in sqNSCLC and other solid tumor indications. Clinical trial information: NCT05359445 .

Modern immunotherapy in the form of T‑cell-based CD19-targeted approaches, such as the approved bispecific T‑cell engager (BiTE antibody) blinatumomab and chimeric antigen receptor T cells (CAR T cells) with the approved products tisagenlecleucel and brexucabtagene autoleucel has revolutionized the treatment of B‑precursor acute lymphoblastic leukemia (ALL). The pivotal clinical trials for the approval, including follow-up studies as well as the now available real-world data outside of these trials, showed that CAR-T cell therapy provides an effective treatment option for patients with relapsed and refractory B‑precursor ALL after chemotherapy, improving an otherwise poor prognosis. In addition to the therapeutic response, side effects of CAR-T cell therapy and their clinical management are discussed. Furthermore, resistance mechanisms are discussed and an outlook on further development is given. The T‑precursor ALL remains a challenge due to its immunological complexity but new developments in CAR-T cell treatment approaches targeting CD5 and CD7 show that progress is also being made in this area.

PURPOSE:Epidermal growth factor receptor (EGFR) mutations are a main actionable driver in non-small cell lung cancer (NSCLC). However, the clinical significance of catenin beta-1 (CTNNB1) comutations remains unclear. This study evaluated outcomes of patients with EGFR/CTNNB1 comutated NSCLC in a dual-center cohort. METHODS:A retrospective analysis of 1,804 patients with NSCLC undergoing next-generation sequencing (NGS) in 2019-2024 at University Hospital Würzburg (single-center cohort, including 15 patients with EGFR/CTNNB1 comutations) was complemented with patients with EGFR/CTNNB1 comutated NSCLC receiving first-line osimertinib at the Thoraxklinik Heidelberg (n = 11) to extend and validate initial findings. We assessed clinical outcomes after first-line osimertinib therapy in 90 EGFR-mutated patients with CTNNB1 wild-type (wt) status and 23 with CTNNB1 comutation. RESULTS:CTNNB1 mutations were identified in 2.0% (36/1,804) of all patients with NSCLC from the single-center cohort, with 41.7% of these also harboring EGFR mutations. Among EGFR-mutant tumors, 7.7% (15/195) exhibited concurrent CTNNB1 mutations. In the dual-center cohort, the objective response rate with first-line osimertinib was 74.4% in CTNNB1-wt (n = 90) and 65.0% in CTNNB1-mutant patients (n = 23; P = .38). Notably, CTNNB1 mutations were associated with significantly longer progression-free survival (PFS; hazard ratio [HR], 0.32; P < .001) and overall survival (OS; HR, 0.33; P = .003). Multivariate analysis confirmed CTNNB1 comutation as an independent prognostic factor for improved PFS (HR, 0.31 [95% CI, 0.14 to 0.69]; P = .004) and OS (HR, 0.26 [95% CI, 0.10 to 0.65]; P = .004). Additionally, CTNNB1 mutations correlated with lower PD-L1 expression (P = .001) and TP53-wt status (P < .001). CONCLUSION:CTNNB1 comutations are associated with lower PD-L1 expression and TP53-wt status, correlating with improved outcomes in patients with EGFR-mutant NSCLC undergoing osimertinib therapy. These results suggest that CTNNB1 comutations may serve as a favorable prognostic biomarker in patients with EGFR-mutant NSCLC. Additional prospective studies are warranted to validate these results.

In contrast to chimeric antigen receptor T cells, T cell receptor (TCR)-engineered T cells can target intracellular tumor-associated antigens crucial for treating solid tumors. However, most trials published so far show limited clinical activity. Here we report interim data from a first-in-human, multicenter, open-label, 3 + 3 dose-escalation/de-escalation phase 1 trial studying IMA203, an autologous preferentially expressed antigen in melanoma (PRAME)-directed TCR T cell therapy in HLA-A*02+ patients with PRAME+ recurrent and/or refractory solid tumors, including melanoma and sarcoma. Primary objectives include the evaluation of safety and tolerability and the determination of the maximum tolerated dose (MTD) and/or recommended dose for extension. Secondary objectives include the evaluation of IMA203 TCR-engineered T cell persistence in peripheral blood, tumor response as well as duration of response. A total of 27 patients were enrolled in the phase 1a dose escalation and 13 patients in the phase 1b dose extension. IMA203 T cells were safe, and the MTD was not reached. Of the 41 patients receiving treatment (that is, who started lymphodepletion), severe cytokine release syndrome was observed in 4.9% (2/41), and severe neurotoxicity did not occur. In the 40 patients treated with IMA203, an overall response rate consisting of patients with unconfirmed or confirmed response (u/cORR) of 52.5% (21/40) and a cORR of 28.9% (11/38) was observed with a median duration of response of 4.4 months (range, 2.4-23.0, 95% confidence interval: 2.6-not reached) across multiple indications. Rapid T cell engraftment and long-term persistence of IMA203 T cells were observed. IMA203 T cells trafficked to all organs, and confirmed responses were more frequent in patients with higher dose. T cell exhaustion was not observed in the periphery; deep responses were enriched at higher PRAME expression; and higher T cell infiltration resulted in longer progression-free survival. Overall, IMA203 showed promising anti-tumor activity in multiple solid tumors, including refractory melanoma. ClinicalTrials.gov identifier: NCT03686124 .

Die moderne Immuntherapie in Form von T‑Zell-basierten, CD19-gerichteten Ansätzen wie dem zugelassenen bispezifischen T‑Zell-Engager (BiTE-Antikörper) Blinatumomab und den CAR-T-Zellen (CAR chimärer Antigenrezeptor) mit den zugelassenen Produkten Tisagenlecleucel und Brexucabtagen-Autoleucel hat die Behandlung der B‑Vorläufer-ALL revolutioniert (ALL akute lymphoblastische Leukämie). Die für die Zulassung entscheidenden klinischen Studien einschließlich der Follow-up-Studien sowie mittlerweile verfügbare Real-world-Daten außerhalb dieser Studien zeigen, dass die CAR-T-Zell-Therapie eine wirksame Behandlungsoption für Patienten mit nach Chemotherapie rezidivierter bzw. chemotherapierefraktärer B‑Vorläufer-ALL bietet und so eine ansonsten schlechte Prognose verbessert. Neben dem therapeutischen Ansprechen werden im vorliegenden Beitrag auch Nebenwirkungen der CAR-T-Zell-Therapie und ihr klinisches Management thematisiert. Darüber hinaus werden Resistenzmechanismen erörtert und ein Ausblick auf die weitere Entwicklung gegeben. Die T‑Vorläufer-ALL bleibt aufgrund ihrer immunologischen Komplexität eine Herausforderung, allerdings zeigen neue Entwicklungen von CD5- und CD7-gerichteten CAR-T-Zell-Therapieansätzen, dass auch hier zunehmend Fortschritte erzielt werden.

2508 Background: Frequent recurrence and limited long-term survival in unresected or metastatic melanoma after relapse from 1L checkpoint inhibitor treatment highlight the critical need for new therapies that deliver deeper, more durable responses. ACTengine IMA203 is an autologous TCR-T targeting PRAME, an intracellular protein displayed as peptide antigen at high density on the surface of multiple solid tumors, including melanoma. Methods: Patients treated in this ongoing Ph1a/b trial (NCT03686124) are ≥18yo, HLA-A*02:01+, PRAME+, have recurrent and/or refractory solid tumors with no additional standard of care treatments available, measurable disease (RECIST1.1) and ECOG PS 0-1. Patients receive Cy/Flu (500 mg/m 2 & 30 mg/m 2 x4 d) lymphodepletion prior to infusion, followed by low-dose IL-2 for 10 days. Results: As of Aug 23, 2024: 70 heavily pretreated patients with solid tumors (median 3 prior systemic therapies) across all dose levels (median total infused dose 2.09x10 9 TCR-T cells (0.08-10.02x10 9 )) were enrolled and assessed for safety. Baseline tumor burden (median sum of diameter): 11.78 cm; LDH > 1 x ULN: 64% of patients. IMA203 had an overall favorable tolerability profile. Most common TEAEs: chemotherapy-related cytopenias (100%), mild to moderate CRS (G1-2: 83%, G3: 11%), infrequent ICANS (G1: 6%, G2: 4%, G3: 4%), no G5 events. Objective responses were observed in melanoma, ovarian cancer, synovial sarcoma, and other tumor types. Successful trafficking of IMA203 cells to various organs was evidenced by their ability to shrink metastatic tumor lesions in the lung, liver, pleura, peritoneum, skin, lymph nodes, adrenal gland, bladder, kidney, spleen, and muscle. Across patients treated in dose escalation and dose expansion, higher doses of IMA203 TCR-T cells were associated with a higher rate of confirmed responses (p = 0.018), whereas tolerability profile remained favorable. Exposure data (C max , AUC) demonstrated a clear dose-dependent improvement in clinical efficacy: Patients with confirmed PR had a higher concentration of IMA203 TCR-T cells in the periphery, compared to patients with unconfirmed PR, SD, and PD. In heavily pretreated patients (median 2 prior systemic therapies) with melanoma at RP2D (1-10x10 9 ) in Ph1b, cORR was 54% (14/26), with tumor shrinkage in 88% (23/26) of patients. Median DOR was 12.1 months with 7/14 confirmed responses ongoing (longest > 2 years). Median PFS was 6 months and median OS not reached at 8.6 months mFU. Updated data with longer follow-up will be presented. Conclusions: IMA203 TCR-T was well tolerated and showed durable objective responses in patients with advanced melanoma. Given its promising risk/benefit profile and high PRAME prevalence in melanoma, a registration-directed Phase 3 trial (SUPRAME; NCT06743126) is underway to further evaluate its efficacy in patients with previously treated (2L) advanced cutaneous melanoma. Clinical trial information: NCT03686124 .

This case report describes the efficacy of selpercatinib, a selective RET inhibitor, in an unusual case of large-cell neuroendocrine pancreatic carcinoma (LCNEPAC) harboring a CCDC6::RET fusion. A 56-year-old male with a history of multiple lines of systemic therapies exhibited marked clinical amelioration shortly after initiating selpercatinib within the LOXO-RET-17001 study (ClinicalTrials.gov ID: NCT03157128, first posted: 2017-05-17). Data from the patient's smartwatch suggested early efficacy before conventional methods, such as serum tumor markers and CT imaging confirmed the antitumor activity. This case not only underscores the efficacy of selpercatinib in treating RET fusion-positive rare tumors but also highlights the potential of wearable technology in cancer care. In conclusion, the standard readings from commercially available wearable devices can be useful for the monitoring of treatment response to targeted therapy and may serve as digital biomarkers in clinical trials. This approach marks a significant advancement in patient-centric healthcare, leveraging technology to enhance the effectiveness and precision of treatment evaluation.

ABSTRACTPurposeOffering equal Patient Access to Precision Oncology (PO) is a major challenge of clinical oncologists and cancer center representatives. Here, we provide an easily transferable model adopted from strategic management science to assess the geographic impact of a cancer center – in terms of general cancer care and PO participation.MethodsAs members of the German WERA alliance, the cancer centers Würzburg, Erlangen, Regensburg and Augsburg merged care data regarding their geographical impact. Specifically, we examined the provenance of patients from WERA’s molecular tumor boards (MTBs) between 2020 and 2022 (n = 2243). As second dimension, we added the provenance of patients receiving general cancer care (termed Total Cancer Care, TCC) by WERA. Clustering our outreach along these two dimensions allowed us to set up a four-quadrant matrix consisting of postal code areas with referrals towards WERA. These areas were re-identified on a map of the Federal State of Bavaria and surrounding regions.ResultsIn terms of positive MTB and general cancer care referrals, the WERA Matrix overlooked an active screening area of n = 821 postal code areas – representing about 50% of Bavaria’s spatial expansion and more than six million inhabitants. The WERA Matrix identified regions successfully connected to our outreach structures in terms of subsidiarity – with general cancer care mainly performed locally but PO performed in cooperation with WERA. At the same time, we detected postal code areas with a potential PO backlog – characterized by high levels of cancer care performed by WERA and low levels or no MTB representation.ConclusionsThe WERA Matrix provided a transparent portfolio of postal code areas, which helped assessing the geographical impact of our PO program. We believe that its intuitive principle can easily be transferred to other cancer centers.

The majority of blood malignancies is incurable and has unforeseeable remitting-relapsing paths in response to different treatments. Cynaropicrin, a natural sesquiterpene lactone from the edible parts of the artichoke plant, has gained increased attention as a chemotherapeutic agent. In this study, we investigated the effects of cynaropicrin against multiple myeloma (MM) cells in vitro and assessed its in vivo effectiveness in a xenograft tumor zebrafish model. We showed that cynaropicrin exerted potent cytotoxicity against a panel of nine MM cell lines and two leukemia cell lines with AMO1 being the most sensitive cell line (IC50 = 1.8 ± 0.3 µM). Cynaropicrin (0.8, 1.9, 3.6 µM) dose-dependently reduced c-Myc expression and transcriptional activity in AMO1 cells that was associated with significant downregulation of STAT3, AKT, and ERK1/2. Cell cycle analysis showed that cynaropicrin treatment arrested AMO1 cells in the G2M phase along with an increase in the sub-G0G1 phase after 24 h. With prolonged treatment times, cells accumulated more in the sub-G0G1 phase, implying cell death. Using confocal microscopy, we revealed that cynaropicrin disrupted the microtubule network in U2OS cells stably expressing α-tubulin-GFP. Furthermore, we revealed that cynaropicrin promoted DNA damage in AMO1 cells leading to PAR polymer production by PARP1 hyperactivation, resulting in AIF translocation from the mitochondria to the nucleus and subsequently to a novel form of cell death, parthanatos. Finally, we demonstrated that cynaropicrin (5, 10 µM) significantly reduced tumor growth in a T-cell acute lymphoblastic leukemia (T-ALL) xenograft zebrafish model. Taken together, these results demonstrate that cynaropicrin causes potent inhibition of hematopoietic tumor cells in vitro and in vivo.

The majority of hematopoietic cancers in adults are incurable and exhibit unpredictable remitting-relapsing patterns in response to various therapies. The proto-oncogene c-MYC has been associated with tumorigenesis, especially in hematological neoplasms. Therefore, targeting c-MYC is crucial to find effective, novel treatments for blood malignancies. To date, there are no clinically approved c-MYC inhibitors. In this study, we virtually screened 1578 Food and Drug Administration (FDA)-approved drugs from the ZINC15 database against c-MYC. The top 117 compounds from PyRx-based screening with the best binding affinities to c-MYC were subjected to molecular docking studies with AutoDock 4.2.6. Retinoids consist of synthetic and natural vitamin A derivatives. All-trans-retinoic acid (ATRA) were highly effective in hematological malignancies. In this study, adapalene, a third-generation retinoid usually used to treat acne vulgaris, was selected as a potent c-MYC inhibitor as it robustly bound to c-MYC with a lowest binding energy (LBE) of −7.27 kcal/mol, a predicted inhibition constant (pKi) of 4.69 µM, and a dissociation constant (Kd value) of 3.05 µM. Thus, we examined its impact on multiple myeloma (MM) cells in vitro and evaluated its efficiency in vivo using a xenograft tumor zebrafish model. We demonstrated that adapalene exerted substantial cytotoxicity against a panel of nine MM and two leukemic cell lines, with AMO1 cells being the most susceptible one (IC50 = 1.76 ± 0.39 µM) and, hence, the focus of this work. Adapalene (0.5 × IC50, 1 × IC50, 2 × IC50) decreased c-MYC expression and transcriptional activity in AMO1 cells in a dose-dependent manner. An examination of the cell cycle revealed that adapalene halted the cells in the G2/M phase and increased the portion of cells in the sub-G0/G1 phase after 48 and 72 h, indicating that cells failed to initiate mitosis, and consequently, cell death was triggered. Adapalene also increased the number of p-H3(Ser10) positive AMO1 cells, which is a further proof of its ability to prevent mitotic exit. Confocal imaging demonstrated that adapalene destroyed the tubulin network of U2OS cells stably transfected with a cDNA coding for α-tubulin-GFP, refraining the migration of malignant cells. Furthermore, adapalene induced DNA damage in AMO1 cells. It also induced apoptosis and autophagy, as demonstrated by flow cytometry and western blotting. Finally, adapalene impeded tumor growth in a xenograft tumor zebrafish model. In summary, the discovery of the vitamin A derivative adapalene as a c-MYC inhibitor reveals its potential as an avant-garde treatment for MM.

The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent.

Supplementary Table 1 Figure S1A, S1B

Supplementary Data from Results from a First-in-Human Phase I Study of Siremadlin (HDM201) in Patients with Advanced Wild-Type TP53 Solid Tumors and Acute Leukemia