A Novel Class of Ribosome Modulating Agents Exploits Cancer Ribosome Heterogeneity to Selectively Target the CMS2 Subtype of Colorectal Cancer.

Ribosomes in cancer cells accumulate numerous patient-specific structural and functional modifications that facilitate tumor progression by modifying protein translation. We have taken a unique synthetic chemistry approach to generate novel macrolides, Ribosome modulating agents (RMA), that are proposed to act distal to catalytic sites and exploit cancer ribosome heterogeneity. The RMA ZKN-157 shows two levels of selectivity: (i) selective translation inhibition of a subset of proteins enriched for components of the ribosome and protein translation machinery that are upregulated by MYC; and (ii) selective inhibition of proliferation of a subset of colorectal cancer cell lines. Mechanistically, the selective ribosome targeting in sensitive cells triggered cell-cycle arrest and apoptosis. Consequently, in colorectal cancer, sensitivity to ZKN-157 in cell lines and patient-derived organoids was restricted to the consensus molecular subtype 2 (CMS2) subtype that is distinguished by high MYC and WNT pathway activity. ZKN-157 showed efficacy as single agent and, the potency and efficacy of ZKN-157 synergized with clinically approved DNA-intercalating agents which have previously been shown to inhibit ribogenesis as well. ZKN-157 thus represents a new class of ribosome modulators that display cancer selectivity through specific ribosome inhibition in the CMS2 subtype of colorectal cancer potentially targeting MYC-driven addiction to high protein translation. Significance: This study demonstrates that ribosome heterogeneity in cancer can be exploited to develop selective ribogenesis inhibitors. The colorectal cancer CMS2 subtype, with a high unmet need for therapeutics, shows vulnerability to our novel selective ribosome modulator. The mechanism suggests that other cancer subtypes with high MYC activation could also be targeted.

Novel read through agent: ZKN-0013 demonstrates efficacy in APCmin model of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a precancerous, colorectal disease characterized by hundreds to thousands of adenomatous polyps caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). Approximately 30% of these mutations are premature termination codons (PTC), resulting in the production of a truncated, dysfunctional APC protein. Consequently, the ß-catenin degradation complex fails to form in the cytoplasm, leading to elevated nuclear levels of ß-catenin and unregulated ß-catenin/wnt-pathway signaling. We present in vitro and in vivo data demonstrating that the novel macrolide, ZKN-0013, promotes read through of premature stop codons, leading to functional restoration of full-length APC protein. Human colorectal carcinoma SW403 and SW1417 cells harboring PTC mutations in the APC gene showed reduced levels of nuclear ß-catenin and c-myc upon treatment with ZKN-0013, indicating that the macrolide-mediated read through of premature stop codons produced bioactive APC protein and inhibited the ß-catenin/wnt-pathway. In a mouse model of adenomatous polyposis coli, treatment of APCmin mice with ZKN-0013 caused a significant decrease in intestinal polyps, adenomas, and associated anemia, resulting in increased survival. Immunohistochemistry revealed decreased nuclear ß-catenin staining in the epithelial cells of the polyps in ZKN-0013-treated APCmin mice, confirming the impact on the ß-catenin/wnt-pathway. These results indicate that ZKN-0013 may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene. KEY MESSAGES: • ZKN-0013 inhibited the growth of human colon carcinoma cells with APC nonsense mutations. • ZKN-0013 promoted read through of premature stop codons in the APC gene. • In APCmin mice, ZKN-0013 treatment reduced intestinal polyps and their progression to adenomas. • ZKN-0013 treatment in APCmin mice resulted in reduced anemia and increased survival.

Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans.

BACKGROUND: Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived ß cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high ß cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. METHODS: Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. RESULTS: Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic ß cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. CONCLUSIONS: In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to ß cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and ß cell function and leads to increased inflammatory signaling.

Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion.

BACKGROUND: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. METHODS: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. RESULTS: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. CONCLUSIONS: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.

Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

A microarray-based antibiotic screen identifies a regulatory role for supercoiling in the osmotic stress response of Escherichia coli.

Changes in DNA supercoiling are induced by a wide range of environmental stresses in Escherichia coli, but the physiological significance of these responses remains unclear. We now demonstrate that an increase in negative supercoiling is necessary for transcriptional activation of a large subset of osmotic stress-response genes. Using a microarray-based approach, we have characterized supercoiling-dependent gene transcription by expression profiling under conditions of high salt, in conjunction with the microbial antibiotics novobiocin, pefloxacin, and chloramphenicol. Algorithmic clustering and statistical measures for gauging cellular function show that this subset is enriched for genes critical in osmoprotectant transport/synthesis and rpoS-driven stationary phase adaptation. Transcription factor binding site analysis also supports regulation by the global stress sigma factor rpoS. In addition, these studies implicate 60 uncharacterized genes in the osmotic stress regulon, and offer evidence for a broader role for supercoiling in the control of stress-induced transcription.