Despite an increasing number of approved therapies, multiple myeloma (MM) remains an incurable disease and only a small number of patients achieve prolonged disease control. Some genes have been linked with response to commonly used anti-MM compounds, including immunomodulators (IMiDs) and proteasome inhibitors (PIs). In this manuscript, we demonstrate an increased incidence of acquired proteasomal subunit mutations in relapsed MM compared to newly diagnosed disease, underpinning a potential role of point mutations in the clonal evolution of MM. Furthermore, we are first to present and functionally characterize four somatic PSMB5 mutations from primary MM cells identified in a patient under prolonged proteasome inhibition, with three of them affecting the PI-binding pocket S1. We confirm resistance induction through missense mutations not only to Bortezomib, but also, in variable extent, to the next-generation PIs Carfilzomib and Ixazomib. In addition, a negative impact on the proteasome activity is assessed, providing a potential explanation for later therapy-induced eradication of the affected tumor subclones in this patient.

Long Non-Coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. Several lncRNAs are involved in cell proliferation and are deregulated in several human tumors. Few lncRNAs have been described to play a role in Acute Lymphoblastic Leukemia (ALL). In this study, we carried out a genome wide lncRNA expression profiling in ALL samples and peripheral blood samples obtained from healthy donors. We detected 43 lncRNAs that were aberrantly expressed in ALL. Interestingly, among them, linc-PINT showed a significant downregulation in T and B-ALL. Re-expression of linc-PINT in ALL cells induced inhibition of leukemic cell growth that was associated with apoptosis induction and cell cycle arrest in G2/M phase. linc-PINT induced the transcription of HMOX1 which reduced the viability of ALL cells. Intriguingly, we observed that treatment with anti-tumoral epigenetic drugs like LBH-589 (Panobinostat) and Curcumin induced the expression of linc-PINT and HMOX1 in ALL. These results indicate that the downregulation of linc-PINT plays a relevant role in the pathogenesis of ALL, and linc-PINT re-expression may be one of the mechanisms exerted by epigenetic drugs to reduce cell proliferation in ALL.

Long non-coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. LncRNAs are as diverse as mRNAs and they normally share the same biosynthetic machinery based on RNA polymerase II, splicing and polyadenylation. However, lncRNAs have low coding potential. Compared to mRNAs, lncRNAs are preferentially nuclear, more tissue specific and expressed at lower levels. Most of the lncRNAs described to date modulate the expression of specific genes by guiding chromatin remodelling factors; inducing chromosomal loopings; affecting transcription, splicing, translation or mRNA stability; or serving as scaffolds for the organization of cellular structures. They can function in cis, cotranscriptionally, or in trans, acting as decoys, scaffolds or guides. These functions seem essential to allow cell differentiation and growth. In fact, many lncRNAs have been shown to exert oncogenic or tumor suppressor properties in several cancers including haematological malignancies. In this review, we summarize what is known about lncRNAs, the mechanisms for their regulation in cancer and their role in leukemogenesis, lymphomagenesis and hematopoiesis. Furthermore, we discuss the potential of lncRNAs in diagnosis, prognosis and therapy in cancer, with special attention to haematological malignancies.

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.

The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.

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Background: Various treatment regimen in multiple myeloma (MM) are based on proteasome inhibition (PI). Although effective at therapy start, most patients relapse and develop drug resistance over time. To better understand the molecular underpinnings associated with PI resistance, we studied genetic and epigenetic alterations of the 26S proteasome genes in PI exposed patients. Methods: We performed a meta-analysis comprised of M3P targeted sequencing datasets and other publicly available WGS/WES sets. A selection of most frequently found mutations was tested in vitro regarding their impact on PI response. DNA promoter methylation of a subset of proteasome genes was determined by targeted Deep Bisulfite Sequencing (DBS), followed up on expression (Taqman qPCR) and validated at functional level (dual-luciferase reporter assay system). Results: The meta-analysis was conducted for a total of 1,752 MM cases, with 1,241 newly diagnosed (NDMM) and 511 progressed MM (PMM) samples. We identified mutations in 32 proteasome genes, with increased incidence from NDMM (6.1% of the patients had mutations in one or more genes) to 10.2% at PMM. Besides PSMB5 encoding for the inhibitor binding site, mainly 19S subunit genes were mutated in areas that impact the recognition of ubiquitinated proteins (PSMD1 and PSMD2), are involved in protein unfolding or gate opening (PSMC1-6). We stably expressed proteasome subunit components bearing frequently observed patient-derived mutations in RPMI-8226 MM cells. All mutants (PSMC6 R242Q, PSMD1 E824K, PSMD1 A887T, PSMD2 M646I, PSMC2 Y429S) displayed an impaired PI response towards Bortezomib (Figure A), Carfilzomib and Ixazomib. Of note, in a fluorescent based, in house clonal competition assay, Bortezomib resistant PSMB5 A20T mutants were outcompeted by WT cells when the drug was removed from co-culture, demonstrating a survival disadvantage through the mutation itself, when no selective pressure of proteasome inhibition was applied. In general, somatic mutations on single gene level were relatively rare, PSMD1, our best candidate gene, was mutated in only 2% of the analyzed MM patients, the remaining genes even to an lower extent, but the proteasome as a whole structure, was frequently affected by mutations. Therefore, we hypothesized that clonal evolution might also act by selecting epigenetic alterations. To address this, we analyzed promoter methylation of PSMC2, PSMC5, PSMC6, PSMD1 and PSMD5 in 42 MM patients by DBS. For PSMD5, NDMM patients and PBMCs were nearly unmethylated (mean methylation: 2.0%±0.020 and 2.0%±0.026), but PMM displayed noticeably increased hypermethylation (6%±0.099). We demonstrated epigenetic silencing by promoter hypermethylation (methylation degree ≥15%) in 20% of PI resistant patients. Moreover, at RNA level we confirmed that patients with high methylation had low expression of PSMD5 and vice versa. To explore the regulatory impact of PSMD5 promoter methylation on gene regulation, we now cloned our amplicon into the backbone of a CpG-free vector (pCpGL). The reporter vector with either the methylated or the unmethylated insert was co-transfected with a Renilla control vector into L363 cells. Luciferase activity of the unmethylated PSMD5 construct was 8 times increased compared to the methylated vector and the controls (vector without insert and non-transfected cells) (Figure B), confirming gene regulation through methylation of this gene. PSMD5, encodes a chaperon recently characterized as the main regulator of 19S proteasome assembly. Gene silencing may represent an adaptive way of cancer cells to bypass proteasome inhibition and escape PI induced proteolytic toxicity by increasing their protein turnover capacity. Conclusion: Altogether our data give evidence that after PI exposition, MM patients harbor acquired regulatory DNA mutations as well as epimutations that affect different proteasomal subunits and, by different modes of action, compromise proteasome activity to escape PI therapy. Figure Disclosures Bittrich: German Research Foundation (DFG): Other: N/A; University of Würzburg: Other: N/A; Bristol Myers Squibb: Research Funding; Otsuka Pharmaceuticals Europe: Other: N/A; Pfizer: Other: Travel Funding; Wilhelm Sander Foundation: Research Funding; Else Kröner Fresenius Foundation: Research Funding; Celgene: Other: Travel Funding, Research Funding; JAZZ Pharmaceuticals: Other: Travel Funding; AMGEN: Other: Travel Funding; University Hospital Wuerzburg: Employment; SANOFI Aventis: Membership on an entity's Board of Directors or advisory committees, N/A, Research Funding.