Cereblon (CRBN), a target of immunomodulatory drugs (IMiD), forms the CRL4 CRBN E3 ubiquitin ligase (CRL4) complex with DDB1, CUL4B and ROC1. 1,2 Under the influence of IMiD, CRL4 polyubiquitinates and thus depletes the transcription factors IKZF1 and IKZF3, resulting in cytotoxicity to multiple myeloma (MM) cells. In vitro, CRBN and IKZF1/3 mutations affecting the CRBN-lenalidomide binding site (degron) cause drug resistance to IMiD. [3][4][5] We hypothesized that mutations in the other components of the CRL4 complex and its targets, Ikaros and Aiolos, likewise interfere with ubiquitin ligase activity, thus contributing to resistance to IMiD. In order to select the most promising patient-derived candidate mutations for functional validation, we first generated a comprehensive overview of point mutations

Combined MEK-BRAF inhibition is a well-established treatment strategy in BRAF-mutated cancer, most prominently in malignant melanoma with durable responses being achieved through this targeted therapy. However, a subset of patients face primary unresponsiveness despite presence of the activating mutation at position V600E, and others acquire resistance under treatment. Underlying resistance mechanisms are largely unknown, and diagnostic tests to predict tumor response to BRAF-MEK inhibitor treatment are unavailable.Multiple myeloma represents the second most common hematologic malignancy, and point mutations in BRAF are detectable in about 10% of patients. Targeted inhibition has been successfully applied, with mixed responses observed in a substantial subset of patients mirroring the widespread spatial heterogeneity in this genomically complex disease. Central nervous system (CNS) involvement is an extremely rare, extramedullary form of multiple myeloma that can be diagnosed in less than 1% of patients. It is considered an ultimate high-risk feature, associated with unfavorable cytogenetics, and, even with intense treatment applied, survival is short, reaching less than 12 months in most cases. Here we not only describe the first patient with an extramedullary CNS relapse responding to targeted dabrafenib and trametinib treatment, we furthermore provide evidence that a point mutation within the capicua transcriptional repressor (CIC) gene mediated the acquired resistance in this patient. The Oncologist 2020;25:112-118 KEY POINTS• BRAF mutations constitute an attractive druggable target in multiple myeloma. This is the first genomic dissection of the central nervous system involvement in a multiple myeloma patient harboring a druggable BRAF V600E mutation. Deep genomic characterization of the extramedullary lesion prompted a personalized therapeutic approach. • Acquisition of CIC mutation confers a mechanism of BRAF-MEK inhibitor drug resistance in multiple myeloma.• The in silico interrogation of the CoMMpass clinical study revealed 10 patients with somatic mutations of CIC and its downregulation at gene expression level in multiple myeloma. • CIC gene silencing decreases the sensitivity of multiple myeloma cells to BRAF-MEK inhibition in vitro. The correlation between CIC downregulation and ETV4/5 nuclear factor expression in multiple myeloma BRAF-mutant cells is shown for the first time. • CIC mutation, its downregulation, and the related downstream effect on MMP24 support disseminative potential providing new clues in the extramedullary biology definition. PATIENT HISTORYAn 81-year-old patient with κ light chain multiple myeloma (MM) was referred to our center after having a seizure and increasing M-proteins. MM had been diagnosed 2 years before and the patient had undergone nine cycles of bortezomibbased combination therapy (VMP) resulting in an initial good disease control. Magnetic resonance imaging of the brain and Correspondence: K.

Cereblon is the direct binding target of the immunomodulatory drugs that are commonly used to treat Multiple Myeloma, the second most frequent hematologic malignancy. Patients respond well to initial IMiD treatment but virtually all develop drug resistance over time with the underlying mechanisms poorly understood. We identified a yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found increased in healthy plasma cells, but more pronounced in IMiD refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNTMi in vitro experiments induced CRBN enhancer demethylation and sensitizing effects on Lenalidomide treatment were observed in two MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in Multiple Myeloma and may predict IMiD response prior treatment.

Recent advances in molecular diagnostics have shown that lesions affecting both copies of the gene for tumor suppressor protein 53 (TP53) count among the most powerful predictors for high-risk disease in multiple myeloma (MM). However, the functional relevance and potential therapeutic implications of single hits to TP53 remain less well understood. Here, we have for the first time approximated the different constellations of mono- and bi-allelic TP53 lesions observed in MM patients within the frame of a single MM cell line model and assessed their potential to disrupt p53 system functionality and to impart drug resistance. Both types of common first hit: point mutation with expression of mutant p53 protein or complete loss of contribution from one of two wildtype alleles strongly impaired p53 system functionality and increased resistance to melphalan. Second hits abolished remaining p53 activity and increased resistance to genotoxic drugs even further. These results fit well with the clinical drive to TP53 single- and double-hit disease in MM patients, provide a rationale for the most commonly observed double-hit constellation (del17p+ TP53 point mutation), and underscore the potential increases in MM cell malignancy associated with any type of initial TP53 lesion.

Multiple myeloma (MM) plasma cell (MMPC) interactions with the microenvironment control MMPC growth, survival, drug-resistance and intra- and extramedullary dissemination. Dissemination of MMPCs through bone marrow niches and in extra-medullary sites is an active process of invasion involving bone marrow endothelial cells, multiple adhesion molecules and chemokine receptors. Since enhanced angiogenesis characterizes MM, we investigated whether junctional adhesion molecule-A (JAM-A) mediated interactions between MM bone marrow endothelial cells (MMECs) and MMPCs impact disease progression. To this end, we analyzed JAM-A expression levels in MMECs of 312 MM patients in two independent cohorts with flow cytometry, namely 111 newly diagnosed (NDMM) and 201 relapsed/refractory (RRMM) and compared them to 36 monoclonal gammopathy of undetermined significance (MGUS) and healthy subjects. To corroborate our data and investigate at a gene-expression level the prognostic value of deregulated genes (FDR<0.1 & P<0.05) we used a Cox-regression model in the CoMMpass dataset (n=326, IA13 release). The role of JAM-A was evaluated by shRNA knockdown and an anti-JAM-A blocking monoclonal antibody. Subsequently, we functionally validated the JAM-A downstream pathways related to cytoskeleton rearrangement, cell proliferation, epithelial mesenchymal transition, invasion and MM dissemination in vitro and in vivo. Surface protein expression of JAM-A on MMECs predicted poor overall survival (OS) in NDMM (not reached (NR) vs. 78 months univariate hazards ratio-HR=9.14, 95% CI 2.8-29.76, P<0.0001) and RRMM patients (NR vs. 130 months, HR=2.96, 95% CI 1.37-6.37, P=0.006) with significant impact also in the progression free survival (PFS) in the advanced stage cohort (8.3 vs. 27 months, HR=1.41, 95% CI 1.05-1.88; P=0.019). A sub-analysis on our cohort with extramedullary disease (EMD) MM revealed that the median OS decreased significantly in patients with JAM-Ahighvs. those with JAM-Alow MMEC expression: 84.1 months vs. not reached, irrespective from the EMD status (log-rank=4.19, P=0.04). Strikingly, among NDMM, these results maintained their significance also in the multivariate analysis (HR=9.11, 95% CI 2.79-29.76, P<0.001); within the RRMM cohort the multivariate analyses confirmed JAM-Ahigh MMECs as a statistically significant independent risk factor for short OS (HR=2.39, 95% CI 1.09-5.28, P=0.03) in much the same way were the Revised international staging system (R-ISS) stages such as stage II (HR=5.34, 95% CI 1.24-22.97, P=0.024), stage III (HR=6.57, 95% CI 1.25-34.54, P=0.026) and chronic kidney disease (HR=2.21, 95% CI 1.06-4.62, P=0.034). Cox stratified model implemented for PFS confirmed only JAM-Ahigh MMECs as a statistically significant risk factor (HR=1.35, 95% CI 1.00-1.81, P=0.044) stratified by chronic kidney disease. Notably, also transcriptional upregulation of JAM-A (RNA-Seq data, CoMMpass) corroborated the prognostic impact (log-rank=13, P=0.0003) and revealed a unique gene-expression signature of activated epithelial-mesenchymal-transition, mTOR/PI3K and focal adhesion pathways in high-risk MM patients with EMD and JAM-Ahigh. Ensuing functional shRNA knockdown of JAM-A reduced MM invasion (P<0.002), angiogenesis (P<0.0001), cell dissemination and migration (P<0.002), cell survival (P<0.001) and expression of cellular-adhesion system molecules such as integrin-beta-1, fibronectin, RAC1 and RHOA (P<0.001). Notably, adding recombinant JAM-A to MMECs enhanced angiogenesis while this was impaired by blocking JAM-A with a specific monoclonal antibody in functional 2D and 3D chorioallantoic membrane-assay and two in vivo MM mouse models. Conclusively, in vivo experiments corroborated our findings that JAM-A blocking halted angiogenesis and reduced MM progression. Collectively, our findings pinpoint JAM-A as a key player propagating a vicious cycle of MMECs and MMPCs interaction; the expression of JAM-A and the related adhesion pathways can prognostically stratify patients in the late disease stages impacting two main MM progression processes: angiogenesis and extra-medullary dissemination. Therefore, we propose JAM-A as a promising MM biomarker and novel therapeutic target in advanced disease. Disclosures Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Rosenwald:MorphoSys: Consultancy. Cavo:celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.