Report of consensus panel 7 from the 11th international workshop on Waldenström macroglobulinemia on priorities for novel clinical trials.

Recent advances in the understanding of Waldenström macroglobulinemia (WM) biology have impacted the development of effective novel agents and improved our knowledge of how the genomic background of WM may influence selection of therapy. Consensus Panel 7 (CP7) of the 11th International Workshop on WM was convened to examine the current generation of completed and ongoing clinical trials involving novel agents, consider updated data on WM genomics, and make recommendations on the design and prioritization of future clinical trials. CP7 considers limited duration and novel-novel agent combinations to be the priority for the next generation of clinical trials. Evaluation of MYD88, CXCR4 and TP53 at baseline in the context of clinical trials is crucial. The common chemoimmunotherapy backbones, bendamustine-rituximab (BR) and dexamethasone, rituximab and cyclophosphamide (DRC), may be considered standard-of-care for the frontline comparative studies. Key unanswered questions include the definition of frailty in WM; the importance of attaining a very good partial response or better (≥VGPR), within stipulated time frame, in determining survival outcomes; and the optimal treatment of WM populations with special needs.

The roles of homologous recombination and the immune system in the genomic evolution of cancer.

A variety of factors, whether extracellular (mutagens/carcinogens and viruses in the environment, chronic inflammation and radiation associated with the environment and/or electronic devices/machines) and/or intracellular (oxidative metabolites of food, oxidative stress due to inflammation, acid production, replication stress, DNA replication/repair errors, and certain hormones, cytokines, growth factors), pose a constant threat to the genomic integrity of a living cell. However, in the normal cellular environment multiple biological pathways including DNA repair, cell cycle, apoptosis and the immune system work in a precise, regulated (tightly controlled), timely and concerted manner to ensure genomic integrity, stability and proper functioning of a cell. If damage to DNA takes place, it is efficiently and accurately repaired by the DNA repair systems. Homologous recombination (HR) which utilizes either a homologous chromosome (in G1 phase) or a sister chromatid (in G2) as a template to repair the damage, is known to be the most precise repair system. HR in G2 which utilizes a sister chromatid as a template is also called an error free repair system. If DNA damage in a cell is so extensive that it overwhelms the repair system/s, the cell is eliminated by apoptosis. Thus, multiple pathways ensure that genome of a cell is intact and stable. However, constant exposure to DNA damage and/or dysregulation of DNA repair mechanism/s poses a risk of mutation and cancer. Oncogenesis, which seems to be a multistep process, is associated with acquisition of a number of genomic changes that enable a normal cell to progress from benign to malignant transformation. Transformed/cancer cells are recognized and killed by the immune system. However, the ongoing acquisition of new genomic changes enables cancer cells to survive/escape immune attack, evolve into a more aggressive phenotype, and eventually develop resistance to therapy. Although DNA repair (especially the HR) and the immune system play unique roles in preserving genomic integrity of a cell, they can also contribute to DNA damage, genomic instability and oncogenesis. The purpose of this article is to highlight the roles of DNA repair (especially HR) and the immune system in genomic evolution, with special focus on gastrointestinal cancer.

MiR-29b antagonizes the pro-inflammatory tumor-promoting activity of multiple myeloma-educated dendritic cells.

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients' adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM.

Protein arginine methyltransferase 5 has prognostic relevance and is a druggable target in multiple myeloma.

Arginine methyltransferases critically regulate cellular homeostasis by modulating the functional outcome of their substrates. The protein arginine methyltransferase 5 (PRMT5) is an enzyme involved in growth and survival pathways promoting tumorigenesis. However, little is known about the biologic function of PRMT5 and its therapeutic potential in multiple myeloma (MM). In the present study, we identified and validated PRMT5 as a new therapeutic target in MM. PRMT5 is overexpressed in patient MM cells and associated with decreased progression-free survival and overall survival. Either genetic knockdown or pharmacological inhibition of PRMT5 with the inhibitor EPZ015666 significantly inhibited growth of both cell lines and patient MM cells. Furthermore, PRMT5 inhibition abrogated NF-κB signaling. Interestingly, mass spectrometry identified a tripartite motif-containing protein 21 TRIM21 as a new PRMT5-partner, and we delineated a TRIM21-dependent mechanism of NF-κB inhibition. Importantly, oral administration of EPZ015666 significantly decreased MM growth in a humanized murine model of MM. These data both demonstrate the oncogenic role and prognostic relevance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome.

Identification and characterization of HLA-A24-specific XBP1, CD138 (Syndecan-1) and CS1 (SLAMF7) peptides inducing antigens-specific memory cytotoxic T lymphocytes targeting multiple myeloma.

X-box binding protein 1 (XBP1), CD138 (Syndecan-1) and CS1 (SLAMF7) are highly expressed antigens in cancers including multiple myeloma (MM). Here, we identify and characterize immunogenic HLA-A24 peptides derived from these antigens for potential vaccination therapy of HLA-A24+ patients with MM. The identified immunogenic HLA-A24-specific XBP1 unspliced (UN)185-193 (I S P W I L A V L), XBP1 spliced (SP)223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F) and CS1240-248 (L F V L G L F L W) peptides induced antigen-specific CTL with anti-MM activity in an HLA-A24 restricted manner. Furthermore, a cocktail containing the four HLA-A24 peptides evoked MM-specific CTL with distinct phenotypic profiles (CD28, CD40L, 41BB, CD38, CD69) and anti-tumor activities, evidenced by perforin upregulation, CD107a degranulation (cytotoxicity) and Th1-type cytokines (IFN-γ/IL-2/TNF-α) production in response to HLA-A24+ MM cells. The multipeptide-specific CTL included antigen-specific memory CD8+ T cells expressing both T-cell activation (CD38, CD69) and immune checkpoints antigens (CTLA, PD-1, LAG-3, TIM-3). These results provide the framework for a multipeptide vaccination therapy to induce tumor-specific CTL in HLA-A24-positive patients with myeloma and other cancers expressing these antigens.

Nucleotide excision repair is a potential therapeutic target in multiple myeloma.

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.

A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma.

Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma.

miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

SAR650984 directly induces multiple myeloma cell death via lysosomal-associated and apoptotic pathways, which is further enhanced by pomalidomide.

The anti-CD38 monoclonal antibody SAR650984 (SAR) is showing promising clinical activity in treatment of relapsed and refractory multiple myeloma (MM). Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define molecular mechanisms of SAR-directed MM cell death and enhanced anti-MM activity triggered by SAR with Pomalidomide (Pom). Without Fc-cross-linking agents or effector cells, SAR specifically induces homotypic aggregation (HA)-associated cell death in MM cells dependent on the level of cell surface CD38 expression, actin cytoskeleton and membrane lipid raft. SAR and its F(ab)'2 fragments trigger caspase 3/7-dependent apoptosis in MM cells highly expressing CD38, even with p53 mutation. Importantly, SAR specifically induces lysosome-dependent cell death (LCD) by enlarging lysosomes and increasing lysosomal membrane permeabilization associated with leakage of cathepsin B and LAMP-1, regardless of the presence of interleukin-6 or bone marrow stromal cells. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR further upregulates reactive oxygen species. Pom enhances SAR-induced direct and indirect killing even in MM cells resistant to Pom/Len. Taken together, SAR is the first therapeutic monoclonal antibody mediating direct cytotoxicity against MM cells via multiple mechanisms of action. Our data show that Pom augments both direct and effector cell-mediated MM cytotoxicity of SAR, providing the framework for combination clinical trials.

Targeting IL-17A in multiple myeloma: a potential novel therapeutic approach in myeloma.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM-BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Rational combination treatment with histone deacetylase inhibitors and immunomodulatory drugs in multiple myeloma.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.

Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity in vitro and in vivo.

Interferon regulatory factor 4 (IRF4) is an attractive therapeutic target in multiple myeloma (MM). We here report that expression of IRF4 mRNA inversely correlates with microRNA (miR)-125b in MM patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive expression of miR-125b-5p by lentiviral vectors or transfection with synthetic mimics impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells in vitro. Apoptotic and autophagy-associated cell death were triggered in MM cells on miR-125b-5p ectopic expression. Importantly, we found that the anti-MM activity of miR-125b-5p was mediated via direct downregulation of IRF4 and its downstream effector BLIMP-1. Moreover, inhibition of IRF4 translated into downregulation of c-Myc, caspase-10 and cFlip, relevant IRF4-downstream effectors. Finally, in vivo intra-tumor or systemic delivery of formulated miR-125b-5p mimics against human MM xenografts in severe combined immunodeficient/non-obese diabetic mice induced significant anti-tumor activity and prolonged survival. Taken together, our findings provide evidence that miR-125b, differently from other hematologic malignancies, has tumor-suppressor activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials.

Multimodality imaging and clinical features in Castleman disease: single institute experience in 30 patients.

OBJECTIVE: To analyse imaging features of subtypes of Castleman disease (CD), emphasizing differentiating features from lymphoma. METHODS: Institutional review board-approved, Health Insurance Portability and Accountability Act compliant, retrospective study examined 30 patients with CD. 30 patients (females, 20; mean age, 46 years; range, 22-87 years) with histopathologically confirmed CD and pre-treatment imaging formed the analytic cohort. Imaging at presentation in all patients [CT, 30; positron emission tomography (PET)/CT, 5; MR, 4; ultrasound, 3] and subsequent imaging in three cases that developed lymphoma was reviewed by two radiologists in consensus. RESULTS: Subtypes: hyaline-vascular (n = 18); multicentric not otherwise specified (NOS) (n = 6); human herpesvirus 8 associated (n = 2); mixed unicentric (n = 2); pure plasma-cell variant (n = 1); and unicentric NOS (n = 1). Distribution: unicentric (n = 17); and multicentric (n = 13). Nodal sites-unicentric: 13 thoracic, 3 abdominal and 1 cervical; multicentric: 9 abdominal, 8 thoracic, 6 cervical, 5 inguinal, 4 axillary and 4 supraclavicular. On CT, differentiating features from lymphoma were calcification (n = 8; 26.7%) and heterogeneous enhancement (n = 5; 19.2%). No association between CD subtype, degree or enhancement pattern, or calcification was noted. On PET/CT (n = 5), nodes were typically fluorine-18 fludeoxyglucose avid (n = 4). On ultrasound (n = 3), nodes were hypoechoic, homogeneous with posterior acoustic enhancement. On MR (n = 4), nodes were hypointense (n = 2) to isointense (n = 2) on T1 weighted images and isointense (n = 1) to hyperintense (n = 3) on T2 weighted images. All (n = 4) demonstrated homogeneous enhancement. Three cases developed non-Hodgkin's lymphoma, two of the three had larger spleens, and these cases had effusions/ascites. CONCLUSION: CD can be unicentric or multicentric and involve nodes above and below the diaphragm. Patients with CD can develop lymphoma. ADVANCES IN KNOWLEDGE: Assessing individual risk of developing lymphoma in patients with CD is difficult, although the findings of splenomegaly, pleural effusion and ascites may be suggestive.

A multiepitope of XBP1, CD138 and CS1 peptides induces myeloma-specific cytotoxic T lymphocytes in T cells of smoldering myeloma patients.

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV) and native CS1239-247 (SLFVLGLFL), for their ability to elicit multipeptide-specific cytotoxic T lymphocytes (MP-CTLs) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTLs generated from SMM patients' T cells show effective anti-MM responses including CD137 (4-1BB) upregulation, CTL proliferation, interferon-γ production and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically, we observed increased total CD3(+)CD8(+) T cells (>80%) and cellular activation (CD69(+)) within the memory SMM MP-CTL (CD45RO(+)/CD3(+)CD8(+)) subset after repeated multipeptide stimulation. Importantly, SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and antitumor activity. In high responders, the effector memory (CCR7(-)CD45RO(+)/CD3(+)CD8(+)) T-cell subset was enriched, whereas the remaining responders' CTL contained a higher frequency of the terminal effector (CCR7(-)CD45RO(-)/CD3(+)CD8(+)) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore, our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease.

CSNK1α1 mediates malignant plasma cell survival.

Here we report that targeting casein kinase 1-α1 (CSNK1α1) is a potential novel treatment strategy in multiple myeloma (MM) therapy distinct from proteasome inhibition. CSNK1α1 is expressed in all the tested MM cell lines and patient MM cells, and is not altered during bortezomib-triggered cytotoxicity. Inhibition of CSNK1α1 kinase activity in MM cells with targeted therapy D4476 or small hairpin RNAs triggers cell G0/G1-phase arrest, prolonged G2/M phase and apoptosis. D4476 also induced cytotoxicity in bortezomib-resistant MM cells and enhanced bortezomib-triggered cytotoxicity. CSNK1α1 signaling pathways include CDKN1B, P53 and FADD; gene signatures involved included interferon-α, tumor necrosis factor-α and LIN9. In addition, reduction of Csnk1α1 prevents cMYC/KRAS12V transformation of BaF3 cells independent of interleukin-3. Impartially, reducing Csnk1α1 prevented development of cMYC/KRAS12V-induced plasmacytomas in mice, suggesting that CSNK1α1 may be involved in MM initiation and progression. Our data suggest that targeting CSNK1α1, alone or combined with bortezomib, is a potential novel therapeutic strategy in MM. Moreover, inhibition of CSNK1α1 may prevent the progression of monoclonal gammopathy of undetermined significance to MM.

Gene expression profile alone is inadequate in predicting complete response in multiple myeloma.

With advent of several treatment options in multiple myeloma (MM), a selection of effective regimen has become an important issue. Use of gene expression profile (GEP) is considered an important tool in predicting outcome; however, it is unclear whether such genomic analysis alone can adequately predict therapeutic response. We evaluated the ability of GEP to predict complete response (CR) in MM. GEP from pretreatment MM cells from 136 uniformly treated MM patients with response data on an IFM, France led study were analyzed. To evaluate variability in predictive power due to microarray platform or treatment types, additional data sets from three different studies (n=511) were analyzed using same methods. We used several machine learning methods to derive a prediction model using training and test subsets of the original four data sets. Among all methods employed for GEP-based CR predictive capability, we got accuracy range of 56-78% in test data sets and no significant difference with regard to GEP platforms, treatment regimens or in newly diagnosed or relapsed patients. Importantly, permuted P-value showed no statistically significant CR predictive information in GEP data. This analysis suggests that GEP-based signature has limited power to predict CR in MM, highlighting the need to develop comprehensive predictive model using integrated genomics approach.

CRM1 inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular mechanisms and therapeutic implications.

The key nuclear export protein CRM1/XPO1 may represent a promising novel therapeutic target in human multiple myeloma (MM). Here we showed that chromosome region maintenance 1 (CRM1) is highly expressed in patients with MM, plasma cell leukemia cells and increased in patient cells resistant to bortezomib treatment. CRM1 expression also correlates with increased lytic bone and shorter survival. Importantly, CRM1 knockdown inhibits MM cell viability. Novel, oral, irreversible selective inhibitors of nuclear export (SINEs) targeting CRM1 (KPT-185, KPT-330) induce cytotoxicity against MM cells (ED50<200 nM), alone and cocultured with bone marrow stromal cells (BMSCs) or osteoclasts (OC). SINEs trigger nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins followed by growth arrest and apoptosis in MM cells. They further block c-myc, Mcl-1, and nuclear factor κB (NF-κB) activity. SINEs induce proteasome-dependent CRM1 protein degradation; concurrently, they upregulate CRM1, p53-targeted, apoptosis-related, anti-inflammatory and stress-related gene transcripts in MM cells. In SCID mice with diffuse human MM bone lesions, SINEs show strong anti-MM activity, inhibit MM-induced bone lysis and prolong survival. Moreover, SINEs directly impair osteoclastogenesis and bone resorption via blockade of RANKL-induced NF-κB and NFATc1, with minimal impact on osteoblasts and BMSCs. These results support clinical development of SINE CRM1 antagonists to improve patient outcome in MM.