Proteasome inhibition is highly effective as a treatment for multiple myeloma, and recently carfilzomib was granted US FDA approval for the treatment of relapsed and refractory multiple myeloma. Here, we report the X-ray crystal structure of the human constitutive 20S proteasome with and without carfilzomib bound at 2.9 and 2.6 Å, respectively. Our data indicate that the S3 and S4 binding pockets play a pivotal role in carfilzomib's selectivity for chymotrypsin-like sites. Structural comparison with the mouse immunoproteasome crystal structure reveals amino acid substitutions that explain carfilzomib's slight preference for chymotrypsin-like subunits of constitutive proteasomes. In addition, comparison of the human proteasome:carfilzomib complex with the mouse proteasome:PR-957 complex reveals new details that explain why PR-957 is selective for immunoproteasomes. Together, the data presented here support the design of inhibitors for either constitutive or immunoproteasomes, with implications for the treatment of cancers as well as autoimmune and neurodegenerative diseases.

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The agreement here with the analytical results is substantially as good as that given by the first structure, and the molecular weight found is somewhat closer to the calculated value in this case.The analysis of the solid polymeric residue left after heating the solid reaction products under reduced pressure, to sublime away any ammonium chloride, while not as conclusive as that presented for the silazanes, led to results approaching the requirements of the simple empirical formula, (Cl-Si^N)*. One such sample, for example, gave the following data:

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Fluorescent in situ hybridization (FISH) is commonly used in the multiple myeloma field to subtype and risk-stratify patients. There are many benefits to FISH based assays, which are widely used around the world and represent true single cell assays. However, there are significant discrepancies in the specific assays, utilization of reflex testing strategies, and enumeration requirements between clinical centers. By comparison next-generation sequencing tests can be designed to simultaneously detect the copy number abnormalities and translocations detected by clinical FISH along with gene mutations that cannot be detected by FISH. As part of the MMRF CoMMpass Study we have compared the results attained using clinical FISH assays compared to sequencing based FISH (Seq-FISH) results. Clinical FISH reports from a random subset of 339 CoMMpass patients were extraction by a single individual based on the ISCN result lines of each report. To validate the accuracy of the central data extraction, two independent cross validations of 10% of the cohort were performed, after which our data entry error rate is expected to be less than 0.348%. The Seq-FISH results were extracted from the whole genome sequencing data available from each patient using a rapid and fully automated informatics process and the results were cross-validated using the matching exome sequencing data for copy number abnormalities and by RNA sequencing data for dysregulated immunoglobulin translocation target genes. There were 230 patients with clinical FISH and Seq-FISH results. In this cohort, 151 translocations were identified by Seq-FISH. This includes translocations to MYC, CCND2, MAFA, and those involving IgK and IgL, which are not tested by clinical FISH. After filtering non-tested translocations there are 118 translocations identified by Seq-FISH. Only 97 of these translocations had a clinical FISH assay performed with 89 (91.75%) of these being detected by clinical FISH, yet spiked target gene expression was observed in all 89 cases by RNA sequencing. Conversely, 93 translocations were called by clinical FISH, of these 89 were called by Seq-FISH(95.7%). Of the 4 translocations only called by clinical FISH, 3 were t(4;14) and 1 was a t(11;14). In two of these t(4;14) cases we did observe spiked target gene expression by RNA sequencing, suggesting these are false negatives by Seq-FISH. However, the remaining two events appear to be false positive clinical FISH results. The t(4;14) event was only observed in 1/200 cells and a co-occuring t(11;14) was also called, which was confirmed by Seq-FISH and spiked gene expression. Similarly, the one t(11;14) was observed in 3/56 cells but a del13q14 was seen in 47/50 cells, unfortunately RNA sequencing data is not available to cross-validate in this case. Plasma cell enrichment or identification is commonly used to prepare myeloma samples for FISH because even in myeloma, the total plasma cell percentage can be low (median 8.3% in the MMRF CoMMpass Baseline Cohort). Therefore, performing FISH on a sample without performing purification or plasma cell identification will indiscriminately assay non-plasma cells and limit the efficacy of the assay. We looked at the two most common translocations in myeloma, t(4;14) and t(11;14), to test the effect of enrichment on sensitivity. Sensitivity was higher for both sets of translocations in the enriched cohort. There was 1 false negative in the enriched population, yielding sensitivities of 100% (32/32) and 95%(19/20) for CCND1 and WHSC1 respectively. For those reports that did not indicate enrichment was performed the observed sensitivities were 86.36% (19/22) and 92.86% (13/14). Seq-FISH identified almost all of the translocations called by clinical FISH and simultaneously; it identified 30 translocations missed by clinical FISH. The translocations that were not reported by clinical FISH can be attributed to a mixture of the correct assay not being performed and the translocation being missed even though the assay was performed. We believe that Seq-FISH is a viable alternative to clinical FISH, with similar specificity and greater sensitivity. It is important to note that a single Seq-FISH assay is sufficient to investigate all translocations, while each translocation must be investigated separately with clinical FISH. As such, Seq-FISH obviates the concern that a translocation would be missed because the correct assay was not performed. Disclosures McBride: Instat: Employment.