Fabian Martin Troschel

Purpose: Current research aims to increase treatment efficacy in diffuse large B‑cell lymphoma (DLBCL). Recently, Musashi‑2 (MSI-2) was identified as a negative prognostic marker and potential therapeutic target in DLBCL. Here, we evaluated the effect of the MSI‑2 inhibitor Ro 08-2750 in DLBCL cells. Methods: First, we established the effects of Ro 08-2750 on DLBCL in cell viability and clonogenicity assays. Changes in cell cycle and apoptosis were analyzed flow cytometrically. We then evaluated modifications in DLBCL treatment response when combining radiation treatment or chemotherapy with Ro 08-2750. Following confirmation of a Ro 08-2750-associated increase in radiation-induced DNA damage via a γH2AX assay, an immunoblot-based assay was used to detect changes in radiation-induced DNA damage repair protein activation. To understand Ro 08-2750-induced modifications in gene expression and protein abundance, we performed RT-qPCR, bulk mRNA-sequencing, western blotting, and quantitative mass spectrometry-based proteomic analyses. Results: Ro 08-2750 reduced cell viability and proliferation in DLBCL. Functionally, Ro 08-2750 induced cell cycle arrest in the S phase, where the DNA synthesis rate was attenuated. Additionally, apoptosis was increased. Ro 08-2750 also sensitized DLBCL cell lines to irradiation but not to chemotherapy, and it increased irradiation-induced DNA double-strand breaks. Transcriptome and proteome analyses showed changes in various tumor-associated signaling pathways, primarily associating Ro 08-2750 with compromised DNA repair, cell cycle, and cell division. Conclusion: We identified Ro 08-2750 as a radiosensitizer in DLBCL. Our results suggest that the combination of Ro 08-2750 with radiation therapy may enhance treatment efficacy.

Background: Recent findings implicate the histone deacetylase (HDAC) inhibitor largazole as an inhibitor of Musashi RNA-binding protein function. Here, we assess this interplay and evaluate the relevance of largazole for triple-negative breast cancer (TNBC) progression and resistance to radiotherapy. Methods: Primary patient-derived TNBC cells and cell lines were treated with largazole and cell vitality, proliferation, motility, cell cycle, DNA synthesis as well as repair, and stemness were analyzed via MTT assay, digital holographic microscopy, and flow cytometry. To unravel the connection between largazole treatment and Musashi expression, miR-125b-5p was assessed after largazole treatment, and overexpressed as well as downregulated in TNBC wildtype cells to determine influence on Musashi levels. Targeted mRNA and protein expression analyses were complemented with RNA sequencing data after largazole treatment. Finally, DNA double strand breaks and post-radiogenic survival were quantified using γ-H2AX, 53BP1, and clonogenic assays. Results: Largazole showed reduced metabolic activity in TNBC, but not in non-malignant cultures. Largazole treatment strongly abrogated proliferation, DNA synthesis, cell motility, and induced a cell cycle arrest. Levels of the Musashi proteins were downregulated after largazole treatment via upregulation of the miR-125b-5p. Protein expression and RNA sequencing analysis indicated a loss of cancer stemness-, cell cycle progression-, and DNA repair-associated signaling. Consequently, radiotherapy-induced DNA double strand breaks were increased while post-radiogenic cell survival was decreased in largazole-treated samples. Conclusion: The HDAC inhibitor largazole compromises tumor growth and motility and downregulates the Musashi proteins via the miR-125b-5p in TNBC. Additionally, largazole acts as a radiosensitizer by attenuating DNA repair, therefore supporting therapeutic efficacy.

Introduction: Treatment resistance is a major hurdle in diffuse large B-cell lymphoma (DLBCL) therapy. Here, we assessed the relevance of the Musashi (MSI) RNA-binding protein family for DLBCL treatment efficacy. As important gene expression regulators, these proteins have previously been associated with tumorigenesis, treatment failure, and reduced survival in other malignancies, making them promising candidates for assessment in the context of DLBCL outcome and therapy resistance.Methods: We first leveraged publicly available gene expression studies to determine expression and prognostic relevance of MSI1 and MSI2 in DLBCL. We then characterized MSI2 co-expressed therapy-relevant signaling. After performing MSI2 knockdown experiments we investigated subsequent effects on DLBCL gene expression in vitro using qPCR, Western blot, protein arrays, and flow cytometry. Finally, cell viability assays and clonogenic assessments were used to assess resistance to radiation, vincristine, and doxorubicin chemotherapy.Results: MSI2 was overexpressed and prognostically unfavorable in univariable and multivariable analyses in DLBCL while MSI1 showed very low expression. High MSI2 expression was associated with increased stemness and DNA repair signaling. MSI2 knockdown led to a loss of stemness-associated markers and compromised DNA repair protein activation while increasing radiation-induced DNA double-strand break levels. Cell survival after either radiotherapy, vincristine or doxorubicin chemotherapy was impaired after MSI2 knockdown in follow-up analyses, suggesting a radio- and chemosensitizing effect.Discussion: We propose that MSI2, a prognostic marker, may modulate the susceptibility of DLBCL towards genotoxic therapy. Suppressing MSI2 may hold promise to sensitize DLBCL to DNA-targeted treatment.

Background: Glioblastoma is the most common malignant brain tumor in adults. Even after maximal safe resection and adjuvant chemoradiotherapy, patients normally relapse after a few years or even months. Standard treatment for recurrent glioblastoma is not yet defined, with re-resection, re-irradiation, and systemic therapy playing key roles. Usually, re-irradiation is combined with concurrent chemotherapy, harnessing the radiosensitizing effects of alkylating agents. Methods: A retrospective analysis of 101 patients with recurrent glioblastoma treated with re-irradiation was conducted, evaluating the survival impact of concurrent chemotherapy regimens, as well as prior resection. Patients were subcategorized according to concurrent chemotherapy (temozolomide vs. CCNU vs. combination of both vs. none) and details are given regarding treatment toxicity and patterns of relapse after first- and second-line treatment. Results: Patients were treated with normo-fractionated re-irradiation (with prescription dose of ~40 Gy to the PTV), resulting in a moderate cumulative EQD2 (~100 Gy). The mean overall survival was 11.3 months (33.5 months from initial diagnosis) and mean progression free survival was 9.5 months. Prior resection resulted in increased survival (p < 0.001), especially when gross total resection was achieved. Patients who received concurrent chemotherapy had significantly longer survival vs. no chemotherapy (p < 0.01), with the combination of CCNU and TMZ achieving the best results. Overall survival was significantly better in patients who received the CCNU + TMZ combination at any time during treatment (first or second line) vs. monotherapy only. The treatment of larger volumes (mean PTV size = 112.7 cm³) was safe and did not result in worse prognosis or increased demand for corticosteroids. Overall, the incidence of high-grade toxicity or sequential radionecrosis (5%) was reasonably low and treatment was tolerated well. While second-line chemotherapy did not seem to influence patterns of relapse, patients who received TMZ + CCNU as first-line treatment had a tendency towards better local control with more out-field recurrence. Conclusions: Normo-fractionated re-irradiation appears to be safe and is accompanied by good survival outcomes, even when applied to larger treatment volumes. Patients amenable to undergo re-resection and achieving concurrent systemic therapy with alkylating agents had better OS, especially when gross total resection was possible. Based on existing data and experiences reflected in this analysis, we advocate for a multimodal approach to recurrent glioblastoma with maximal safe re-resection and adjuvant second chemoradiation. The combination of TMZ and CCNU for patients with methylated MGMT promoter yielded the best results in the primary and recurrent situation (together with re-RT). Normo-fractionated RT enables the use of more generous margins and is tolerated well.

Purpose: This study aimed to analyze treatment-related risk factors for sensorineural hearing loss (SNHL) and an indication for hearing aids (IHA) in medulloblastoma patients after craniospinal radiotherapy (CSRT) and platin-based chemotherapy (PCth). Methods: A total of 58 patients (116 ears) with medulloblastoma and clinically non-relevant pre-treatment hearing thresholds were included. Cranial radiotherapy and PCth were applied sequentially according to the HIT 2000 study protocol or post-study recommendations, the NOA-07 protocol, or the PNET (primitive neuroectodermal tumor) 5 MB therapy protocol. Audiological outcomes up to a maximum post-therapeutic follow-up of 4 years were assessed. The incidence, post-treatment progression, and time-to-onset of SNHL, defined as Muenster classification grade ≥MS2b, were evaluated. Risk factors for IHA were analyzed separately. Results: While 39 patients received conventionally fractionated RT (CFRT; group 1), 19 patients received hyperfractionated RT (HFRT; group 2). Over a median follow-up of 40 months, 69.2% of ears in group 1 experienced SNHL ≥MS2b compared to 89.5% in group 2 (p = 0.017). In multivariable Cox regressions analysis, younger age and increased mean cochlear radiation dose calculated as the equivalent dose in 2‑Gy fractions (EQD2) were associated with time-to-onset of SNHL ≥MS2b (p = 0.019 and p = 0.023, respectively) and IHA (p < 0.001 and p = 0.016, respectively). Tomotherapy and supine positioning were associated with a lower risk for IHA in univariable modelling only (p = 0.048 and p = 0.027, respectively). Conclusion: Young age and cochlear EQD2 D_mean ≥40 Gy are significant risk factors for the incidence, degree, and time-to-event of SNHL as well as for IHA in medulloblastoma patients.

Reducing side effects in non-cancerous tissue is a key aim of modern radiotherapy. Here, we assessed whether the use of the antioxidants hydroxytyrosol (HT) and thioredoxin-mimetic peptide CB3 (TMP) attenuated radiation-induced normal tissue toxicity in vitro. We used primary human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (HaCaT) as normal tissue models. Cells were treated with HT and TMP 24 h or immediately prior to irradiation. Reactive oxygen species (ROS) were assessed via luminescent- and fluorescence-based assays, migration was investigated using digital holographic microscopy, and clonogenic survival was quantified by colony formation assays. Angiogenesis and wound healing were evaluated via time-dependent microscopy. Secreted cytokines were validated in quantitative polymerase chain reaction (qPCR) studies. Treatment with HT or TMP was well tolerated by cells. The application of either antioxidant before irradiation resulted in reduced ROS formation and a distinct decrease in cytokines compared to similarly irradiated, but otherwise untreated, controls. Antioxidant treatment also increased post-radiogenic migration and angiogenesis while accelerating wound healing. HT or TMP treatment immediately before radiotherapy increased clonogenic survival after radiotherapy, while treatment 24 h before radiotherapy enhanced baseline proliferation. Both antioxidants may decrease radiation-induced normal tissue toxicity and deserve further pre-clinical investigation.

Purpose: Sarcopenia may complicate treatment in cancer patients. Herein, we assessed whether sarcopenia measurements derived from radiation planning computed tomography (CT) were associated with complications and tumor progression during radiochemotherapy for glioblastoma. Methods: Consecutive patients undergoing radiotherapy planning for glioblastoma between 2010 and 2021 were analyzed. Retrocervical muscle cross-sectional area (CSA) was measured via threshold-based semi-automated radiation planning CT analysis. Patients in the lowest sex-specific quartile of muscle measurements were defined as sarcopenic. We abstracted treatment characteristics and tumor progression from the medical records and performed uni- and multivariable time-to-event analyses. Results: We included 363 patients in our cohort (41.6% female, median age 63 years, median time to progression 7.7 months). Sarcopenic patients were less likely to receive chemotherapy (p < 0.001) and more likely to be treated with hypofractionated radiotherapy (p = 0.005). Despite abbreviated treatment, they more often discontinued radiotherapy (p = 0.023) and were more frequently prescribed corticosteroids (p = 0.014). After treatment, they were more often transferred to inpatient palliative care treatment (p = 0.035). Finally, progression-free survival was substantially shorter in sarcopenic patients in univariable (median 5.1 vs. 8.4 months, p < 0.001) and multivariable modeling (hazard ratio 0.61 [confidence interval 0.46–0.81], p = 0.001). Conclusion: Sarcopenia is a strong risk factor for treatment discontinuation and reduced progression-free survival in glioblastoma patients. We propose that sarcopenic patients should receive intensified supportive care during radiotherapy and during follow-up as well as expedited access to palliative care.