Cancer-associated mutations in the condensin II subunit CAPH2 cause genomic instability through telomere dysfunction and anaphase chromosome bridges.

Genome instability in cancer drives tumor heterogeneity, undermines the success of therapies, and leads to metastasis and recurrence. Condensins are conserved chromatin-binding proteins that promote genomic stability, mainly by ensuring proper condensation of chromatin and mitotic chromosome segregation. Condensin mutations are found in human tumors, but it is not known how or even if such mutations promote cancer progression. In this study, we focus on condensin II subunit CAPH2 and specific CAPH2 mutations reported to be enriched in human cancer patients, and we test how CAPH2 cancer-specific mutations may lead to condensin II complex dysfunction and contribute to genome instability. We find that R551P, R551S, and S556F mutations in CAPH2 cause genomic instability by causing DNA damage, anaphase defects, micronuclei, and chromosomal instability. DNA damage and anaphase defects are caused primarily by ataxia telangiectasia and Rad3-related-dependent telomere dysfunction, as anaphase bridges are enriched for telomeric repeat sequences. We also show that these mutations decrease the binding of CAPH2 to the ATPase subunit SMC4 as well as the rest of the condensin II complex, and decrease the amount of CAPH2 protein bound to chromatin. Thus, in vivo the R551P, R551S, and S556F cancer-specific CAPH2 mutant proteins are likely to impair condensin II complex formation, impede condensin II activity during mitosis and interphase, and promote genetic heterogeneity in cell populations that can lead to clonal outgrowth of cancer cells with highly diverse genotypes.

The novel ß2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells.

Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the ß5 and ß1 activity, but not the ß2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate ß2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the ß2 proteasome activity. LU-102 inhibited the ß2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of ß1 and ß5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, ß2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.

Cell-line-specific high background in the Proteasome-Glo assay of proteasome trypsin-like activity.

Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity.

Phlorins bearing different substituents at the sp3-hybridized meso-position.

Phlorins bearing different substituents at the sp(3)-hybridized meso-position were investigated. The extent to which different substituents at this unique position can influence phlorin spectroscopic properties, structure, and stability is of interest given that such substituents are not in direct conjugation with the phlorin macrocycle. While the effect of various substituents at the sp(2)-hybridized positions has been the subject of prior investigations, the impact of different substituents at the saturated carbon atom has not been systematically examined. In this study, phlorins with different combinations of geminal methyl and phenyl substituents were prepared in yields of 24-49% via dipyrromethane + dipyrromethanedicarbinol routes, and their NMR spectra, UV-vis spectra, X-ray crystal structures, and stability toward light and air were compared. The nature of the substituents at the sp(3)-hybridized position was found to impact spectroscopic properties, structure, and stability to varying degrees. Thus, the choice of substituents at the sp(3)-hybridized meso-position provides a further option for altering phlorin properties.

Human Islet Amyloid Polypeptide (hIAPP) Protofibril-Specific Antibodies for Detection and Treatment of Type 2 Diabetes.

Type 2 diabetes mellitus (T2D) is a major public health concern and is characterized by sustained hyperglycemia due to insulin resistance and destruction of insulin-producing ß cells. One pathological hallmark of T2D is the toxic accumulation of human islet amyloid polypeptide (hIAPP) aggregates. Monomeric hIAPP is a hormone normally co-secreted with insulin. However, increased levels of hIAPP in prediabetic and diabetic patients can lead to the formation of hIAPP protofibrils, which are toxic to ß cells. Current therapies fail to address hIAPP aggregation and current screening modalities do not detect it. Using a stabilizing capping protein, monoclonal antibodies (mAbs) can be developed against a previously nonisolatable form of hIAPP protofibrils, which are protofibril specific and do not engage monomeric hIAPP. Shown here are two candidate mAbs that can detect hIAPP protofibrils in serum and hIAPP deposits in pancreatic islets in a mouse model of rapidly progressing T2D. Treatment of diabetic mice with the mAbs delays disease progression and dramatically increases overall survival. These results demonstrate the potential for using novel hIAPP protofibril-specific mAbs as a diagnostic screening tool for early detection of T2D, as well as therapeutically to preserve ß cell function and target one of the underlying pathological mechanisms of T2D.

Structure-Based Design of Inhibitors Selective for Human Proteasome ß2c or ß2i Subunits.

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the ß1c, ß1i, ß5c, and ß5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting ß2c or ß2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the ß2c- and ß2i-selective compounds LU-002c (4; IC50 ß2c: 8 nM, IC50 ß2i/ß2c: 40-fold) and LU-002i (5; IC50 ß2i: 220 nM, IC50 ß2c/ß2i: 45-fold), respectively. Co-crystal structures with ß2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of ß2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.

Inhibition of the Proteasome ß2 Site Sensitizes Triple-Negative Breast Cancer Cells to ß5 Inhibitors and Suppresses Nrf1 Activation.

The proteasome inhibitors carfilzomib (Cfz) and bortezomib (Btz) are used successfully to treat multiple myeloma, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the ß5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of ß1 and ß2 to demonstrate that inhibiting a second site of the proteasome, particularly the ß2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both ß5 and ß2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual ß5 and ß2 inhibitors for the treatment of solid tumors.