Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.

T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL). CLL cells downregulate T-cell responses by expressing 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 improve responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated mithramycin analogue, MTM ox 32E, to suppress CLL IL-10. We found MTM ox 32E inhibited mouse and human CLL IL-10 production and maintained Tcell effector function. In the Eμ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden while it 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 with anti-PD-L1 alone. Combination therapy also produced more interferon-γ + , cytotoxic effector KLRG1 + , and memory CD8 + T-cells, with fewer exhausted T-cells than ICB alone. Since current therapies for CLL do not target IL-10, this provides a novel strategy to increase the efficacy of T-cell-based immunotherapies.

Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2′-oxime (MTM ox ) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTM ox analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusionindependent/dependent selectivity indices for select 2′-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTM ox 32E (a Phe−Trp dipeptide-based 2′-conjugate) for in vivo testing. Relative to MTM, MTM ox 32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.

T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL). CLL cells downregulate T-cell responses by expressing 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 improve responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated mithramycin analogue, MTMox32E, to suppress CLL IL-10. We found MTMox32E inhibited mouse and human CLL IL-10 production and maintained T-cell effector function. In the Eμ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden while it 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 with anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, with fewer exhausted T-cells than ICB alone. Since current therapies for CLL do not target IL-10, this provides a novel strategy to increase the efficacy of T-cell-based immunotherapies.

Introduction Chromosomal translocations involving the ETS family of transcription factors are common in Ewing sarcoma, prostate cancer, and leukemia. These translocations lead in overexpression of aberrant ETS transcription factors, which drive tumorigenesis. Mithramycin (MTM) was shown to inhibit EWS/FLI1, the most common ETS related transcription factor in Ewing sarcoma, presumably through interference at its DNA binding sites on promoter regions. However, MTM has a short half‐life and a narrow therapeutic window marked by severe liver and hematological toxicities. Considering that MTM has a pKa of 5, we hypothesized that its rapid pharmacokinetic (PK) clearance is attributed to liver uptake by organic anion transporter polypeptides (OATP), which may also contribute to the observed hepatotoxicity. Here, we sought to develop analogues with specificity toward ETS transcription factors and reduced toxicity by attempting to modify the physicochemical properties of MTM. Methods A series of analogues were obtained by modifying the 3‐side chain of MTM to increase its pKa. Specificity toward cells expressing ETS transcription factors was determined by cytotoxicity studies in ETS expressing cancer cells (n=9) vs. cancer cell lines (n=9) lacking ETS. Luciferase reporter constructs were used to indirectly evaluate if MTM analogues affected interaction of EWS/FLI1 and Sp1 binding to promoters. Gene expression was tested by qRT‐PCR and interaction at specific promoter sites was determined by ChIP‐qRT‐PCR. The PK of MTM was determined in the presence and absence of rifampin pretreatment and in OATP1B1/1B3 transgenic mice. Select analogues were tested in vivo to identify the maximum tolerated dose and determine pharmacokinetics (PK). Results Semisynthetic substitutions using aromatic amino acids at the 3‐side chain of MTM lead to a significant increase (0.5–1.5 units) of the pKa. Growth inhibition assays identified several MTM analogues with > 10 fold selectivity toward ETS cell lines and this was corroborated by reporter and expression assays. The clearance of mithramycin was significantly decreased by rifampicin pretreatment and MTM exposure was significantly higher in Oatp1a/1b KO mice expressing OATP1B3 (transgenic) vs KO mice expressing OATP1B1. Select analogues were further tested to determine relative toxicity in mice and were found to be tolerated at 10–30 fold higher doses than MTM. Significantly, the presumed shift in pKa afforded a dramatic difference in pharmacokinetic properties resulting in relative drug exposures (area under the concentration‐time curves) that were 50–400 higher than MTM at their respective maximum tolerated doses. Conclusion These studies show that substitutions at the 3‐side chain of MTM yield analogues that are selectively more cytotoxic against tumor cell lines expressing aberrant ETS transcription factors. Further, significantly altered pharmacokinetics of these analogues may expand the therapeutic window and lead to the development of a new generation of MTM analogues amenable for targete...