Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34CD38) as well as the leukemic bulk (CD34CD38) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

Dual- or multi-target drugs have emerged as a promising alternative to combination therapies. Proteasome inhibitors (PIs) possess synergistic activity with histone deacetylase (HDAC) inhibitors due to the simultaneous blockage of the ubiquitin-degradation and aggresome pathways. Here, we present the design, synthesis, binding modes and anticancer properties of RTS-V5 as the first-in-class dual HDAC-proteasome ligand. The inhibition of both targets was confirmed by biochemical and cellular assays as well as X-ray crystal structures of the 20S proteasome and HDAC6 complexed with RTS-V5. Cytotoxicity assays with leukemia and multiple myeloma cell lines as well as therapy-refractory primary patient-derived leukemia cells demonstrated that RTSV5 possesses potent and selective anticancer activity. Our results will thus guide the structure-based optimization of dual HDAC-proteasome inhibitors for the treatment of hematological malignancies.

Based on gene expression profiles, diffuse large B-cell lymphoma (DLBCL) is subdivided into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 as well as BCL2 copy-number gains. Here, we employ immune phenotyping, RNA sequencing, and wholeexome sequencing to characterize a Myd88-and BCL2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression compared with GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and PD-1 blockade significantly increased the overall survival of lymphoma-bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.SignificAnce: Oncogenic Myd88 and BCL2 cooperate in murine DLBCL lymphomagenesis. The resulting lymphomas display morphologic and transcriptomic features reminiscent of human ABC-DLBCL. Data derived from our Myd88/BCL2-driven autochthonous model demonstrate that combined BCL2 and PD-1 blockade displays substantial preclinical antilymphoma activity, providing preclinical proofof-concept data, which pave the way for clinical translation.

Multitarget drugs are an emerging alternative to combination therapies. In three iterative cycles of design, synthesis, and biological evaluation, we developed a novel type of potent hybrid inhibitors of bromodomain, and extra-terminal (BET) proteins and histone deacetylases (HDACs) based on the BET inhibitor XD14 and well-established HDAC inhibitors. The most promising new hybrids, 49 and 61, displayed submicromolar inhibitory activity against HDAC1−3 and 6, and BRD4(1), and possess potent antileukemia activity. 49 induced apoptosis more effectively than the combination of ricolinostat and birabresib (1:1). The most balanced dual inhibitor, 61, induced significantly more apoptosis than the related control compounds 62 (no BRD4(1) affinity) and 63 (no HDAC inhibition) as well as the 1:1 combination of both. Additionally, 61 was well tolerated in an in vivo zebrafish toxicity model. Overall, our data suggest an advantage of dual HDAC/BET inhibitors over the combination of two single targeted compounds.

Aberrantly activated CD4 T memory cells play a central role in the development of type-1-diabetes. Interleukin-7 promotes generation of autoimmune memory T cells and increased Interleukin-7 availability is associated with type-1-diabetes susceptibility. T-cell-mediated immune pathology at onset of type-1-diabetes is well defined, but characteristics of long-term symptomatic disease stages remain largely elusive. In the present study, memory CD4 T-cell activation and cytokine expression as well as sensitivity to Interleukin-7 in vitro were compared between patients with type-1-diabetes at clinical onset (n=25), long-term symptomatic disease (median duration 4.5 years, n=19) and matched healthy controls (n=21). T-cell responses of type-1-diabetes patients were characterized by higher frequencies of cytokine and activation marker expressing CD4 memory T cells as compared to healthy controls. Notably, correction for individual cytokine expression levels revealed qualitative differences of cytokine profiles characterized by significantly increased TNFα and decreased IL-2-expressing T-cell proportions in long-term type-1-diabetes patients. IL-7-mediated T-cell co-stimulation induced quantitative and qualitative cytokine expression differences highly similar to type-1-diabetes-specific profiles. In addition, CD4 memory T cells from children with long-term type-1-diabetes were more sensitive to in vitro IL-7 co-stimulation. Global transcriptome analysis revealed IL-7 induced expression differences of CD4 T cells, including increased IL-2R expression and effects on subsequent T-cell receptor activation. We conclude that long-term symptomatic type-1-diabetes patients differed in memory T-cell cytokine profiles and Interleukin-7 co-stimulation. Regulation of IL-2 expression and sensitivity are affected with possible consequences for disease course and severity at long-term type-1-diabetes stages.