The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

Effects of Mithramycin on BCL11A Gene Expression and on the Interaction of the BCL11A Transcriptional Complex to γ-Globin Gene Promoter Sequences.

The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in erythroid precursor cells (ErPCs) from ß-thalassemia patients. In this respect, previously published studies indicate that MTH is very active in inducing increased expression of γ-globin genes in erythroid cells. This is clinically relevant, as it is firmly established that HbF induction is a valuable approach for the therapy of ß-thalassemia and for ameliorating the clinical parameters of sickle-cell disease (SCD). Therefore, the identification of MTH biochemical/molecular targets is of great interest. This study is inspired by recent robust evidence indicating that the expression of γ-globin genes is controlled in adult erythroid cells by different transcriptional repressors, including Oct4, MYB, BCL11A, Sp1, KLF3 and others. Among these, BCL11A is very important. In the present paper we report evidence indicating that alterations of BCL11A gene expression and biological functions occur during MTH-mediated erythroid differentiation. Our study demonstrates that one of the mechanisms of action of MTH is a down-regulation of the transcription of the BCL11A gene, while a second mechanism of action is the inhibition of the molecular interactions between the BCL11A complex and specific sequences of the γ-globin gene promoter.

Macrophage-organoid co-culture model for identifying treatment strategies against macrophage-related gemcitabine resistance.

BACKGROUND: Gemcitabine resistance (GR) is a significant clinical challenge in pancreatic adenocarcinoma (PAAD) treatment. Macrophages in the tumor immune-microenvironment are closely related to GR. Uncovering the macrophage-induced GR mechanism could help devise a novel strategy to improve gemcitabine treatment outcomes in PAAD. Therefore, preclinical models accurately replicating patient tumor properties are essential for cancer research and drug development. Patient-derived organoids (PDOs) represent a promising in vitro model for investigating tumor targets, accelerating drug development, and enabling personalized treatment strategies to improve patient outcomes. METHODS: To investigate the effects of macrophage stimulation on GR, co-cultures were set up using PDOs from three PAAD patients with macrophages. To identify signaling factors between macrophages and pancreatic cancer cells (PCCs), a 97-target cytokine array and the TCGA-GTEx database were utilized. The analysis revealed CCL5 and AREG as potential candidates. The role of CCL5 in inducing GR was further investigated using clinical data and tumor sections obtained from 48 PAAD patients over three years, inhibitors, and short hairpin RNA (shRNA). Furthermore, single-cell sequencing data from the GEO database were analyzed to explore the crosstalk between PCCs and macrophages. To overcome GR, inhibitors targeting the macrophage-CCL5-Sp1-AREG feedback loop were evaluated in cell lines, PDOs, and orthotopic mouse models of pancreatic carcinoma. RESULTS: The macrophage-CCL5-Sp1-AREG feedback loop between macrophages and PCCs is responsible for GR. Macrophage-derived CCL5 activates the CCR5/AKT/Sp1/CD44 axis to confer stemness and chemoresistance to PCCs. PCC-derived AREG promotes CCL5 secretion in macrophages through the Hippo-YAP pathway. By targeting the feedback loop, mithramycin improves the outcome of gemcitabine treatment in PAAD. The results from the PDO model were corroborated with cell lines, mouse models, and clinical data. CONCLUSIONS: Our study highlights that the PDO model is a superior choice for preclinical research and precision medicine. The macrophage-CCL5-Sp1-AREG feedback loop confers stemness to PCCs to facilitate gemcitabine resistance by activating the CCR5/AKT/SP1/CD44 pathway. The combination of gemcitabine and mithramycin shows potential as a therapeutic strategy for treating PAAD in cell lines, PDOs, and mouse models.

Abrogation of stemness in osteosarcoma by the mithramycin analog EC-8042 is mediated by its ability to inhibit NOTCH-1 signaling.

Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has demonstrated an efficient ability to eliminate tumor cells, including cancer stem cell subpopulations (CSC), in sarcomas. In transcriptomic and protein expression analyses, we identified NOTCH1 signaling as one of the main pro-stemness pathways repressed by EC-8042 in osteosarcomas. Overexpression of NOTCH-1 resulted in a reduced anti-tumor effect of EC-8042 in CSC-enriched 3D tumorspheres cultures. On the other hand, the depletion of the NOTCH-1 downstream target HES-1 was able to enhance the action of EC-8042 on CSCs. Moreover, HES1 depleted cells failed to recover after treatment withdrawal and showed reduced tumor growth potential in vivo. In contrast, mice xenografted with NOTCH1-overexpressing cells responded worse than parental cells to EC-8042. Finally, we found that active NOTCH1 levels in sarcoma patients was associated to advanced disease and lower survival. Overall, these data highlight the relevant role that NOTCH1 signaling plays in mediating stemness in osteosarcoma. Moreover, we demonstrate that EC-8042 is powerful inhibitor of NOTCH signaling and that the anti-CSC activity of this mithramycin analog highly rely on its ability to repress this pathway.

Iron deficiency downregulates ENPEP to promote angiogenesis in liver tumors.

The abnormal iron metabolism in liver cancer leads to iron deficiency in tumor tissues. We previously found that iron deficiency promoted liver cancer metastasis, but the mechanisms were not fully understood. In the present study, we identified that the angiogenesis-associated glutamyl aminopeptidase (ENPEP) was consistently decreased in iron-deficient liver tissues, iron-deficient liver tumors, and iron-deprived liver cancer cells. Interestingly, the lower expression of ENPEP was correlated with the poor prognosis of liver cancer patients, while the biomarkers of angiogenesis, CD31 and CD34, were increased in tumor tissues. In vivo imaging of liver-orthotopically implanted and tail vein-injected liver cancer cells showed that iron deficiency increased the pulmonary metastasis of liver cancer. The angiogenesis in iron-deficient tumors was enhanced, and the expression of ENPEP was decreased. Silencing ENPEP expression increased the migration of liver cancer cells and the proliferation of cocultured HUVECs. By sequence analysis, we found that the transcription factor SP1 possessed abundant binding sites in the ENPEP promoter region. Its combination with ENPEP promoters was verified by chromatin immunoprecipitation. The inhibition of SP1 by mithramycin A effectively restored the expression of ENPEP, which was decreased by iron deficiency. In conclusion, these results revealed that iron deficiency in liver tumors decreased the expression of ENPEP by SP1 and increased the angiogenesis and metastasis of liver tumors, which further explained the mechanism by which iron deficiency promoted liver cancer metastasis.

Mithplatins: Mithramycin SA-Pt(II) Complex Conjugates for the Treatment of Platinum-Resistant Ovarian Cancers.

DNA coordinating platinum (Pt) containing compounds cisplatin and carboplatin have been used for the treatment of ovarian cancer therapy for four decades. However, recurrent Pt-resistant cancers are a major cause of mortality. To combat Pt-resistant ovarian cancers, we designed and synthesized a conjugate of an anticancer drug mithramycin with a reactive Pt(II) bearing moiety, which we termed mithplatin. The conjugates displayed both the Mg2+ -dependent noncovalent DNA binding characteristic of mithramycin and the covalent crosslinking to DNA of the Pt. The conjugate was three times as potent as cisplatin against ovarian cancer cells. The DNA lesions caused by the conjugate led to the generation of DNA double-strand breaks, as also observed with cisplatin. Nevertheless, the conjugate was highly active against both Pt-sensitive and Pt-resistant ovarian cancer cells. This study paves the way to developing mithplatins to combat Pt-resistant ovarian cancers.

Mithramycin suppresses tumor growth by regulating CD47 and PD-L1 expression.

Mithramycin A (MIT) has reacquired extensive research attention due to its anti-solid tumor activity and improved pharmacological production. Mechanismly, MIT was broadly used as a c-Myc inhibitor, and c-Myc regulated CD47 and PD-L1 expression which has been demonstrated. However, how MIT affects immune check-point molecules remains unknown. In this study, we found CD47 expression was higher in melanoma of pan-tissue array. MIT inhibited CD47 expression both in mRNA and protein level in melanoma cells (SK-MEL-28 and B16). MIT inhibited c-Myc, Sp-1 and CD47 expression in a concentration-dependent way. MIT inhibited the surface CD47 expression and promoted the phagocytosis of SK-MEL-28 cells by THP-1 cells. We found MIT inhibited tumor growth in melanoma allograft mice and CD47 expression in tumor mass. We also found MIT upregulated PD-L1 expression in cancer cells possibly via inhibiting PD-L1 ubiquitination, increasing ROS and IFN-γ. Combination of MIT and anti-PD-1 antibody showed enhanced antitumor activity compared to MIT and anti-PD-1 antibody alone in MC38 allograft mice. Using immune checkpoint array we found MIT inhibited expression of FasL and Galectin3. These results suggest that MIT inhibits CD47 expression, while improves PD-L1 expression. Furthermore, the combination of MIT and anti-PD-1 antibody exerts potent antitumor effect.

Everolimus and plicamycin specifically target chemoresistant colorectal cancer cells of the CMS4 subtype.

Colorectal cancers (CRC) can be classified into four consensus molecular subtypes (CMS), among which CMS1 has the best prognosis, contrasting with CMS4 that has the worst outcome. CMS4 CRC is notoriously resistant against therapeutic interventions, as demonstrated by preclinical studies and retrospective clinical observations. Here, we report the finding that two clinically employed agents, everolimus (EVE) and plicamycin (PLI), efficiently target the prototypic CMS4 cell line MDST8. As compared to the prototypic CMS1 cell line LoVo, MDST8 cells treated with EVE or PLI demonstrated stronger cytostatic and cytotoxic effects, increased signs of apoptosis and autophagy, as well as a more pronounced inhibition of DNA-to-RNA transcription and RNA-to-protein translation. Moreover, nontoxic doses of EVE and PLI induced the shrinkage of MDST8 tumors in mice, yet had only minor tumor growth-reducing effects on LoVo tumors. Altogether, these results suggest that EVE and PLI should be evaluated for their clinical activity against CMS4 CRC.

Mithramycin delivery systems to develop effective therapies in sarcomas.

BACKGROUND: Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an attractive approach to identify effective therapeutic compounds. One example is mithramycin (MTM), a natural antibiotic which has demonstrated a strong antitumour activity in several tumour types, including sarcomas. However, its widespread use in the clinic was limited by its poor toxicity profile. RESULTS: In order to improve the therapeutic index of MTM, we have loaded MTM into newly developed nanocarrier formulations. First, polylactide (PLA) polymeric nanoparticles (NPs) were generated by nanoprecipitation. Also, liposomes (LIP) were prepared by ethanol injection and evaporation solvent method. Finally, MTM-loaded hydrogels (HG) were obtained by passive loading using a urea derivative non-peptidic hydrogelator. MTM-loaded NPs and LIP display optimal hydrodynamic radii between 80 and 105 nm with a very low polydispersity index (PdI) and encapsulation efficiencies (EE) of 92 and 30%, respectively. All formulations show a high stability and different release rates ranging from a fast release in HG (100% after 30 min) to more sustained release from NPs (100% after 24 h) and LIP (40% after 48 h). In vitro assays confirmed that all assayed MTM formulations retain the cytotoxic, anti-invasive and anti-stemness potential of free MTM in models of myxoid liposarcoma, undifferentiated pleomorphic sarcoma and chondrosarcoma. In addition, whole genome transcriptomic analysis evidenced the ability of MTM, both free and encapsulated, to act as a multi-repressor of several tumour-promoting pathways at once. Importantly, the treatment of mice bearing sarcoma xenografts showed that encapsulated MTM exhibited enhanced therapeutic effects and was better tolerated than free MTM. CONCLUSIONS: Overall, these novel formulations may represent an efficient and safer MTM-delivering alternative for sarcoma treatment.

Combination Therapy of Mithramycin A and Immune Checkpoint Inhibitor for the Treatment of Colorectal Cancer in an Orthotopic Murine Model.

The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and blocking this axis has been found to be effective in a subset of patients. Although blocking PD-L1 has been shown to be effective in 5-10% of patients, the majority of the cohorts show resistance to this checkpoint blockade (CB) therapy. Multiple factors assist in the growth of resistance to CB, among which T cell exhaustion and immunosuppressive effects of immune cells in the tumor microenvironment (TME) play a critical role along with other tumor intrinsic factors. We have previously shown the polyketide antibiotic, Mithramycin-A (Mit-A), an effective agent in killing cancer stem cells (CSCs) in vitro and in vivo in a subcutaneous murine model. Since TME plays a pivotal role in CB therapy, we tested the immunomodulatory efficacy of Mit-A with anti-PD-L1 mAb (αPD-L1) combination therapy in an immunocompetent MC38 syngeneic orthotopic CRC mouse model. Tumors and spleens were analyzed by flow cytometry for the distinct immune cell populations affected by the treatment, in addition to RT-PCR for tumor samples. We demonstrated the combination treatment decreases tumor growth, thus increasing the effectiveness of the CB. Mit-A in the presence of αPD-L1 significantly increased CD8+ T cell infiltration and decreased immunosuppressive granulocytic myeloid-derived suppressor cells and anti-inflammatory macrophages in the TME. Our results revealed Mit-A in combination with αPD-L1 has the potential for augmented CB therapy by turning an immunologically "cold" into "hot" TME in CRC.

MYB mediates downregulation of the colorectal cancer metastasis suppressor heterogeneous nuclear ribonucleoprotein L-like during epithelial-mesenchymal transition.

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.

Insight into mithramycin disruption of ETS transcription leads to improved understanding of more selective analogs.

Fusion products with the ETS family of transcription factors play critical roles in the etiology of several cancers. In this issue of Structure, Hou et al. (2020) provide insight into allosteric mechanisms by which mithramycin and its analogs perturb protein-DNA interactions in higher-order complexes at a DNA enhancer site.

Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia.

T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTMox32E) to suppress CLL IL-10. MTMox32E treatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector function in vitro. In the Eµ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8+ T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.

Specific protein 1 inhibitor mithramycin A protects cardiomyocytes from myocardial infarction via interacting with PARP.

Specific protein 1 (SP1) might act as a critical transcription regulator in myocardial infarction (MI), but little evidence about its function in regulating cardiac apoptosis, a major cause of MI development, has been revealed. This study tried to investigate the role of SP1 in MI and its interaction with poly-ADP-ribose polymerase (PARP)-1 by using SP1 inhibitor, mithramycin A (mithA). Primary mouse cardiomyocytes and commercial mouse cardiomyocytes were subjected to mithA treatment under hypoxia conditions, while cell viability, Nix promoter activity, and its expression were detected correspondingly. PARP overexpression and knockdown were conducted, respectively, in mithA-treated and SP1-overexpressing cells. Co-immunoprecipitation was used to verify the interaction between PARP and SP1. For in vivo experiments, mithA administration was performed after the injections of adenovirus for PARP overexpression, and then, MI introduction was carried out. Infarct size and lactate dehydrogenase level were measured to assess MI injury. SP1 inhibitor mithA attenuated hypoxia-induced decrease of cell viability and Nix transcriptional activation, which could be inhibited by PARP overexpression. Knockdown of PARP prevented SP1-induced transcription of Nix and cell viability change, and PARP showed direct interaction with SP1. Furthermore, mithA administration reduced MI injuries, while PARP overexpression could suppress the improvement. The cardioprotective role of SP1 inhibitor mithA was demonstrated here expanding the role of SP1 in MI development involving hypoxia-induced cardiac apoptosis. Moreover, PARP acted as a transcriptional coactivator in Nix transcription involving its interaction with SP1.

Erb­B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53­responsive microRNA­3184­5p in cervical cancer cells.

The oncogenic role of Erb­B2 Receptor Tyrosine Kinase 2 (ERBB2) has been identified in several types of cancer, but less is known on its function and mechanism of action in cervical cancer cells. The present study employed a multipronged approach to investigate the role of ERBB2 in cervical cancer. ERBB2 and microRNA (miR)­3184­5p expression was assessed in patient­derived cervical cancer biopsy tissues, revealing that higher levels of ERBB2 and lower levels of miR­3184­5p were associated with clinicopathological indicators of cervical cancer progression. Furthermore, ERBB2 stimulated proliferation, migration and sphere­formation of cervical cancer cells in vitro. This effect was mediated by enhanced phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit α activity. Additionally, it was revealed that miR­3184­5p directly suppressed ERBB2 in cervical cancer cells. The p53 activator Mithramycin A stimulated p53 and miR­3184­5p expression, thereby lowering the levels of ERBB2 and attenuating proliferation, migration and sphere­formation of cervical cancer cells. In conclusion, the findings of the present study suggested ERBB2 as an oncogenic protein that may promote invasiveness in cervical cancer cells. Treatment of cervical cancer cells with the p53 activator Mithramycin A restored the levels of the endogenous ERBB2 inhibitor miR­3184­5p and may represent a novel treatment strategy for cervical cancer.

Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor.

Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second-generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1-deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM-defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3-day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice.

Allosteric interference in oncogenic FLI1 and ERG transactions by mithramycins.

ETS family transcription factors of ERG and FLI1 play a key role in oncogenesis of prostate cancer and Ewing sarcoma by binding regulatory DNA sites and interfering with function of other factors. Mithramycin (MTM) is an anti-cancer, DNA binding natural product that functions as a potent antagonist of ERG and FLI1 by an unknown mechanism. We present a series of crystal structures of the DNA binding domain (DBD) of ERG/FLI1 culminating in a structure of a high-order complex of the ERG/FLI1 DBD, transcription factor Runx2, core-binding factor beta (Cbfß), and MTM on a DNA enhancer site, along with supporting DNA binding studies using MTM and its analogues. Taken together, these data provide insight into allosteric mechanisms underlying ERG and FLI1 transactions and their disruption by MTM analogues.

Mithramycin A Radiosensitizes EWS:Fli1+ Ewing Sarcoma Cells by Inhibiting Double Strand Break Repair.

PURPOSE: The oncogenic EWS:Fli1 fusion protein is a key transcriptional mediator of Ewing sarcoma initiation, progression, and therapeutic resistance. Mithramycin A (MithA) is a potent and specific inhibitor of transcription mediated by the EWS:Fli1. We tested the hypothesis that pretreatment with MithA could selectively radiosensitize EWS:Fli1+ tumor cells by altering the transcriptional response to radiation injury. METHODS AND MATERIALS: A panel of 4 EWS:Fli1+ and 3 EWS:Fli1- Ewing sarcoma cell lines and 1 nontumor cell line were subjected to MithA dose-response viability assays to determine the relative potency of MithA in cells possessing or lacking the EWS:Fli1 fusion. Radiosensitization by MithA was evaluated by clonogenic survival assays in vitro and in a murine xenograft model. DNA damage was evaluated by comet assay and γ-H2Ax flow cytometry. Immunoblotting, flow cytometry, and reverse-transcription, polymerase chain reaction were used to evaluate DNA damage-induced signaling and repair processes and apoptosis. RESULTS: We found that MithA alone could potently and selectively inhibit the growth of EWS:Fli1+ tumor cells, but not cells lacking this fusion. Pretreatment with MithA for 24 hours before irradiation significantly reduced clonogenic survival in vitro and delayed tumor regrowth in vivo, prolonging survival of EWS:Fli1+ tumor-bearing mice. Although MithA did not increase the level of DNA double-strand breaks, mechanistic studies revealed that MithA pretreatment selectively inhibited DNA double-strand break repair through downregulation of EWS:Fli1-mediated transcription, leading to tumor cell death by apoptosis. CONCLUSIONS: Our data indicate that MithA is an effective radiosensitizer of EWS:Fli1+ tumors and may achieve better local control at lower doses of radiation.

Mithramycin selectively attenuates DNA-damage-induced neuronal cell death.

DNA damage triggers cell death mechanisms contributing to neuronal loss and cognitive decline in neurological disorders, including traumatic brain injury (TBI), and as a side effect of chemotherapy. Mithramycin, which competitively targets chromatin-binding sites of specificity protein 1 (Sp1), was used to examine previously unexplored neuronal cell death regulatory mechanisms via rat primary neurons in vitro and after TBI in mice (males). In primary neurons exposed to DNA-damage-inducing chemotherapy drugs in vitro we showed that DNA breaks sequentially initiate DNA-damage responses, including phosphorylation of ATM, H2AX and tumor protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent intrinsic apoptosis. Mithramycin was highly neuroprotective in DNA-damage-dependent neuronal cell death, inhibiting chemotherapeutic-induced cell death cascades downstream of ATM and p53 phosphorylation/activation but upstream of p53-induced expression of pro-apoptotic molecules. Mithramycin reduced neuronal upregulation of BH3-only proteins and mitochondrial dysfunction, attenuated caspase-3/7 activation and caspase substrates' cleavage, and limited c-Jun activation. Chromatin immunoprecipitation indicated that mithramycin attenuates Sp1 binding to pro-apoptotic gene promoters without altering p53 binding suggesting it acts by removing cofactors required for p53 transactivation. In contrast, the DNA-damage-independent neuronal death models displayed caspase initiation in the absence of p53/BH3 activation and were not protected even when mithramycin reduced caspase activation. Interestingly, experimental TBI triggers a multiplicity of neuronal death mechanisms. Although markers of DNA-damage/p53-dependent intrinsic apoptosis are detected acutely in the injured cortex and are attenuated by mithramycin, these processes may play a reduced role in early neuronal death after TBI, as caspase-dependent mechanisms are repressed in mature neurons while other, mithramycin-resistant mechanisms are active. Our data suggest that Sp1 is required for p53-mediated transactivation of neuronal pro-apoptotic molecules and that mithramycin may attenuate neuronal cell death in conditions predominantly involving DNA-damage-induced p53-dependent intrinsic apoptosis.

Mithramycin suppresses DNA damage repair via targeting androgen receptor in prostate cancer.

The dependency of prostate cancer (PCa) growth on androgen receptor (AR) signaling has been harnessed to develop first-line therapies for high-risk localized and metastatic PCa treatment. However, the occurrence of aberrant expression, mutated or splice variants of AR confers resistance to androgen ablation therapy (ADT), radiotherapy or chemotherapy in AR-positive PCa. Therapeutic strategies that effectively inhibit the expression and/or transcriptional activity of full-length AR, mutated AR and AR splice variants have remained elusive. In this study, we report that mithramycin (MTM), an antineoplastic antibiotic, suppresses cell proliferation and exhibits dual inhibitory effects on expression and transcriptional activity of AR and AR splice variants. MTM blocks AR recruitment to its genomic targets by occupying AR enhancers and causes downregulation of AR target genes, which includes key DNA repair factors in DNA damage repair (DDR). We show that MTM significantly impairs DDR and enhances the effectiveness of ionizing radiation or the radiomimetic agent Bleomycin in PCa. Thus, the combination of MTM treatment with RT or radiomimetic agents, such as bleomycin, may present a novel effective therapeutic strategy for patients with high-risk, clinically localized PCa.