SummaryMultiple myeloma (MM) is a plasma cell neoplasm that presents with a major biological and clinical heterogeneity. We here investigated the spectrum of clonal and subclonal mutations of DIS3, an active part of the exosome complex, that may play a role in the development or progression of MM. The whole coding sequence of DIS3 was subjected to deep sequencing in 81 uniformly-treated MM patients and 12 MM cell lines and the overall occurrence of DIS3 mutations as well as the presence of DIS3 mutations in minor and major subclones were correlated with cytogenetic alterations and clinical parameters. Our study identified DIS3 mutations in 9/81 patients that were associated with 13q14 deletions and IGH translocations on the cytogenetic level. Specifically, we detected seven novel somatic DIS3 single nucleotide variants (SNVs) and defined three hot spot mutations within the RNB domain. Lastly, we found a trend towards a shorter median overall survival for patients with DIS3 mutations, and patients carrying DIS3 mutations in minor subclones of their tumours showed a significantly worse response to therapy compared to patients with DIS3 mutations in the major subclone.

Telomerase represents an attractive target for a mechanism-based therapeutic approach because its activation has been associated with unlimited proliferation in most cancer cells. Recently, a nonnucleosidic small molecule inhibitor, BIBR1532 (2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid), has been identified that is highly selective for inhibition of telomerase, resulting in delayed growth arrest of tumor cells. Here we examined the effects of BIBR1532 in different leukemia cell lines as well as in primary cells from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) in short-term culture assays. We observed a dose-dependent direct cytotoxicity in concentrations ranging from 30 to 80 M. Interestingly, cell death was not dependent on the catalytic activity of telomerase but was delayed in cells with very long telomeres. We observed timedependent individual telomere erosion, which was associated with loss of telomeric repeat binding factor 2 (TRF2) and increased phosphorylation of p53. Importantly, the proliferative capacity of normal CD34 ؉ cells from cord blood and leukapheresis samples was not affected by treatment with BIBR1532. We conclude that using this class of telomerase inhibitor at higher concentrations exerts a direct cytotoxic effect on malignant cells of the hematopoietic system, which appears to derive from direct damage of the structure of individual telomeres and must be dissected from telomerase-suppressed overall telomere shortening. (Blood. 2005; 105:1742-1749)

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Recent advances in genomic sequencing technologies now allow results from deep next-generation sequencing to be obtained within clinically meaningful timeframes, making this an attractive approach to better guide personalized treatment strategies. No multiple myeloma-specific gene panel has been established so far; we therefore designed a 47-gene-targeting gene panel, containing 39 genes known to be mutated in ≥3%of multiple myeloma cases and eight genes in pathways therapeutically targeted in multiple myeloma (MM). We performed targeted sequencing on tumor/germline DNA of 25 MM patients in which we also had a sequential sample post treatment. Mutation analysis revealed KRAS as the most commonly mutated gene (36 % in each time point), followed by NRAS (20 and 16 %), TP53 (16 and 16 %), DIS3 (16 and 16 %), FAM46C (12 and 16 %), and SP140 (12 and 12 %). We successfully tracked clonal evolution and identified mutation acquisition and/or loss in FAM46C, FAT1, KRAS, NRAS, SPEN, PRDM1, NEB, and TP53 as well as two mutations in XBP1, a gene associated with bortezomib resistance. Thus, we present the first longitudinal analysis of a MM-specific targeted sequencing gene panel that can be used for individual tumor characterization and for tracking clonal evolution over time.

Summary We constructed a multiple myeloma (MM)-specific gene panel for targeted sequencing and investigated 72 untreated high-risk (del17p) MM patients. Mutations were identified in 78% of the patients. While the majority of studied genes were mutated at similar frequency to published literature, the prevalence of TP53 mutation was increased (28%) and no mutations were found in FAM46C. This study provides a comprehensive insight into the mutational landscape of del17p high-risk MM. Additionally, our work demonstrates the practical use of a customized sequencing panel, as an easy, cheap and fast approach to characterize the mutational profile of MM.

Lenalidomide is an immunomodulatory compound with high clinical activity in multiple myeloma. Lenalidomide binding to the Cereblon (CRBN) E3 ubiquitin ligase results in targeted ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) leading to growth inhibition of multiple myeloma cells. Recently, Basigin (BSG) was identified as another protein regulated by CRBN that is involved in the activity of lenalidomide. Here, we analyzed the prognostic value of IKZF1, IKZF3, CRBN and BSG mRNA expression levels in pretreatment plasma cells from 60 patients with newly diagnosed multiple myeloma uniformly treated with lenalidomide in combination with intensive chemotherapy within a clinical trial. We found that IKZF1 mRNA expression levels are significantly associated with progression-free survival (PFS). Patients in the lowest quartile (Q1) of IKZF1 expression had a superior PFS compared with patients in the remaining quartiles (Q2-Q4; 3-year PFS of 86 vs 51%, P=0.01). This translated into a significant better overall survival (100 vs 74%, P=0.03). Subgroup analysis revealed a significant impact of IKZF1, IKZF3 and BSG expression levels on PFS in cytogenetically defined standard-risk but not high-risk patients. Our data suggest a prognostic role of IKZF1, IKZF3 and BSG expression levels in lenalidomide-treated multiple myeloma.

The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide are highly effective treatments for multiple myeloma. However, virtually all patients eventually relapse due to acquired drug resistance with resistance-causing genetic alterations being found only in a small subset of cases. To identify non-genetic mechanisms of drug resistance, we here perform integrated global quantitative tandem mass tag (TMT)-based proteomic and phosphoproteomic analyses and RNA sequencing in five paired pre-treatment and relapse samples from multiple myeloma patients. These analyses reveal a CDK6-governed protein resistance signature that includes myeloma high-risk factors such as TRIP13 and RRM1. Overexpression of CDK6 in multiple myeloma cell lines reduces sensitivity to IMiDs while CDK6 inhibition by palbociclib or CDK6 degradation by proteolysis targeting chimeras (PROTACs) is highly synergistic with IMiDs in vitro and in vivo. This work identifies CDK6 upregulation as a druggable target in IMiD-resistant multiple myeloma and highlights the use of proteomic studies to uncover non-genetic resistance mechanisms in cancer.

Introduction: Involvement of the central nervous system in patients with multiple myeloma is a rare event. We evaluated the diagnostic workup and prognosis of patients with leptomeningeal myelomatosis (LMM). Methods: Between April 2005 and April 2016, we identified 16 cases with LMM. The involvement was diagnosed by magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) cytology as well as flow cytometry. Fluorescence in situ hybridization (FISH) was used in 8/16 cases. In 1 case, genome-wide screening for genetic alterations using single-nucleotide polymorphism (SNP) array analysis was performed. Results: The median time from initial diagnosis until the occurrence of LMM was 434 days. At diagnosis, the median age was 60 years. The median cell count was 21/µL (range 1–1,333/µL). All CSF samples showed malignant pleocytosis, confirmed by flow cytometry in 12/16 patients. FISH revealed high-risk features in the majority of samples. Treatment for LMM consisted of intrathecal chemotherapy and radiation therapy. Genome-wide screening assays revealed different subclones. The outcome was dismal with a median overall survival after the diagnosis of LMM of 82 days. Conclusion: By combining several technical procedures, it is possible to identify most patients with LMM. Management of affected patients is challenging and the survival short after a diagnosis of LMM.