Signaling In Multiple Myeloma Research Articles

NT157 exhibits antineoplastic effects by targeting IRS and STAT3/5 signaling in multiple myeloma

Multiple myeloma (MM) is a prevalent hematological malignancy with high recurrence and no definitive cure. The current study revisits the role of the IGF1/IGF1R axis in MM, introducing a novel inhibitor, NT157. The IGF1/IGF1R pathway is pivotal in MM, influencing cell survival, proliferation, and migration and impacting patient survival outcomes. NT157 targets intracellular proteins such as IRS and STAT proteins and demonstrates antineoplastic potential in hematological malignancies and solid tumors. In the present study, we assessed IGF1R signaling-related gene expression in MM patients and healthy donors, unveiling significant distinctions. MM cell lines displayed varying expression patterns of IGF1R-related proteins. A gene dependence analysis indicated the importance of targeting receptor and intracellular elements over autocrine IGF1. NT157 exhibited inhibitory effects on MM cell viability, clonal growth, cell cycle progression, and survival. Moreover, NT157 reduced IRS2 expression and STAT3, STAT5, and RPS6 activation and modulated oncogenes and tumor suppressors, fostering a tumor-suppressive molecular profile. In summary, our study demonstrates that the IGF1/IGF1R/IRS signaling axis is differentially activated in MM cells and the NT157’s capacity to modulate crucial molecular targets, promoting antiproliferative effects and apoptosis in MM cells. NT157 may offer a multifaceted approach to enhance MM therapy.

Interleukin-10 increases macrophage-mediated chemotherapy resistance via FABP5 signaling in multiple myeloma

Macrophages (MΦs) protect multiple myeloma (MM) cells from chemotherapy-induced apoptosis, and interleukin-10 (IL-10) is frequently elevated in the MM microenvironment. However, the role of IL-10 in MΦ-induced tumor chemotherapy resistance has not yet been clarified. In the present study, bone marrow-derived MΦs were treated with IL-10 (IL10-MΦs), and IL10-MΦ-induced MM chemotherapy resistance was evaluated. IL-10 promoted MΦ-mediated resistance to MM chemotherapy. In addition, IL-10 treatment increased lipid accumulation and fatty acid β-oxidation in MΦs. Mechanistically, IL-10 increased fatty acid binding protein 5 (FABP5) expression in MΦs, and targeting FABP5 decreased MM chemotherapy resistance induced by IL10-MΦs in vitro and enhanced chemotherapeutic efficacy in vivo. Inhibition of FABP5 decreased the expression of Carnitine Palmitoyltransferase 1A (CPT1A) in IL10-MΦs. In addition, inhibition of CPT1A in IL10-MΦs decreased IL10-MΦ-mediated MM chemotherapy resistance. Peroxisome proliferator-activated receptor γ (PPARγ) is upstream of FABP5 signaling. Inhibition of PPARγ in IL10-MΦs decreased IL10-MΦ-mediated MM chemotherapy resistance in vitro. Collectively, our work indicates that IL-10 enhances MΦ-mediated MM chemotherapy resistance via FABP5 signaling and targeting FABP5 has potentially important clinical implications.

Magnolol and Temozolomide exhibit a synergistic anti-glioma activity through MGMT inhibition

Temozolomide (TMZ) is the leading chemotherapeutic agent used for glioma therapy due to its good oral absorption and blood-brain barrier permeability. However, its anti-glioma efficacy may be limited due to its adverse effects and resistance development. O6-Methylguanine-DNA-methyltransferase (MGMT), an enzyme associated with TMZ resistance, is activated via the NF-κB pathway, which is found to be upregulated in glioma. TMZ also upregulates NF-κB signaling like many other alkylating agents. Magnolol (MGN), a natural anti-cancer agent, has been reported to inhibit NF-κB signaling in multiple myeloma, cholangiocarcinoma, and hepatocellular carcinoma. MGN has already shown promising results in anti-glioma therapy. However, the synergistic action of TMZ and MGN has not been explored. Therefore, we investigated the effect of TMZ and MGN treatment in glioma and observed their synergistic pro-apoptotic action in both in vitro and in vivo glioma models. To explore the mechanism of this synergistic action, we found that MGN inhibits MGMT enzyme both in vitro and in vivo glioma. Next, we established the link between NF-κB signaling and MGN-induced MGMT inhibition in glioma. MGN inhibits the phosphorylation of p65, a subunit of NF-κB, and its nuclear translocation to block NF-κB pathway activation in glioma. MGN-induced NF-κB inhibition results in the transcriptional inhibition of MGMT in glioma. TMZ and MGN combinatorial treatment also impedes p65 nuclear translocation to inhibit MGMT in glioma. We observed a similar effect of TMZ and MGN treatment in the rodent glioma model. Thus, we concluded that MGN potentiates TMZ-induced apoptosis in glioma by inhibiting NF-κB pathway-mediated MGMT activation.

Ectopic CH60 mediates HAPLN1-induced cell survival signaling in multiple myeloma.

Multiple myeloma (MM), the second most common hematological malignancy, is generally considered incurable because of the development of drug resistance. We previously reported that hyaluronan and proteoglycan link protein 1 (HAPLN1) produced by stromal cells induces activation of NF-κB, a tumor-supportive transcription factor, and promotes drug resistance in MM cells. However, the identity of the cell surface receptor that detects HAPLN1 and thereby engenders pro-tumorigenic signaling in MM cells remains unknown. Here, we performed an unbiased cell surface biotinylation assay and identified chaperonin 60 (CH60) as the direct binding partner of HAPLN1 on MM cells. Cell surface CH60 specifically interacted with TLR4 to evoke HAPLN1-induced NF-κB signaling, transcription of anti-apoptotic genes, and drug resistance in MM cells. Collectively, our findings identify a cell surface CH60-TLR4 complex as a HAPLN1 receptor and a potential molecular target to overcome drug resistance in MM cells.

AATF/Che-1 localizes to paraspeckles and suppresses R-loops accumulation and interferon activation in Multiple Myeloma.

Several kinds of stress promote the formation of three-stranded RNA:DNA hybrids called R-loops. Insufficient clearance of these structures promotes genomic instability and DNA damage, which ultimately contribute to the establishment of cancer phenotypes. Paraspeckle assemblies participate in R-loop resolution and preserve genome stability, however, the main determinants of this mechanism are still unknown. This study finds that in Multiple Myeloma (MM), AATF/Che-1 (Che-1), an RNA-binding protein fundamental to transcription regulation, interacts with paraspeckles via the lncRNA NEAT1_2 (NEAT1) and directly localizes on R-loops. We systematically show that depletion of Che-1 produces a marked accumulation of RNA:DNA hybrids. We provide evidence that such failure to resolve R-loops causes sustained activation of a systemic inflammatory response characterized by an interferon (IFN) gene expression signature. Furthermore, elevated levels of R-loops and of mRNA for paraspeckle genes in patient cells are linearly correlated with Multiple Myeloma progression. Moreover, increased interferon gene expression signature in patients is associated with markedly poor prognosis. Taken together, our study indicates that Che-1/NEAT1 cooperation prevents excessive inflammatory signaling in Multiple Myeloma by facilitating the clearance of R-loops. Further studies on different cancer types are needed to test if this mechanism is ubiquitously conserved and fundamental for cell homeostasis.

Biological Hallmarks and Emerging Strategies to Target STAT3 Signaling in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy, characterized by an abnormal accumulation of plasma cells in the bone marrow. Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription factor that modulates the transcription of multiple genes to regulate various principal biological functions, for example, cell proliferation and survival, stemness, inflammation and immune responses. Aberrant STAT3 activation has been identified as a key driver of tumorigenesis in many types of cancers, including MM. Herein, we summarize the current evidence for the role of STAT3 in affecting cancer hallmark traits by: (1) sustaining MM cell survival and proliferation, (2) regulating tumor microenvironment, (3) inducing immunosuppression. We also provide an update of different strategies for targeting STAT3 in MM with special emphasis on JAK inhibitors that are currently undergoing clinical trials. Finally, we discuss the challenges and future direction of understanding STAT3 signaling in MM biology and the clinical development of STAT3 inhibitors.

Pathogenic signaling in multiple myeloma

Multiple myeloma is a common hematological malignancy of plasma cells, the terminally differentiated B cells that secrete antibodies as part of the adaptive immune response. Significant progress has been made in treating multiple myeloma, but this disease remains largely incurable, and most patients will eventually suffer a relapse of disease that becomes refractory to further therapies. Moreover, a portion of patients with multiple myeloma present with disease that is refractory to all treatments from the initial diagnosis, and no current therapeutic approaches can help. Therefore, the task remains to advance new therapeutic strategies to help these vulnerable patients. One strategy to meet this challenge is to unravel the complex web of pathogenic signaling pathways in malignant plasma cells and use this information to design novel precision medicine strategies to assist these patients most at risk.

The multifunctional role of Notch signaling in multiple myeloma.

Multiple myeloma (MM) is a hematologic cancer characterized by uncontrolled growth of malignant plasma cells in the bone marrow and currently is incurable. The bone marrow microenvironment plays a critical role in MM. MM cells reside in specialized niches where they interact with multiple marrow cell types, transforming the bone/bone marrow compartment into an ideal microenvironment for the migration, proliferation, and survival of MM cells. In addition, MM cells interact with bone cells to stimulate bone destruction and promote the development of bone lesions that rarely heal. In this review, we discuss how Notch signals facilitate the communication between adjacent MM cells and between MM cells and bone/bone marrow cells and shape the microenvironment to favor MM progression and bone disease. We also address the potential and therapeutic approaches used to target Notch signaling in MM.

Cystathionine β Synthase/Hydrogen Sulfide Signaling in Multiple Myeloma Regulates Cell Proliferation and Apoptosis.

Objective-To investigate cystathionine β synthase (CBS)/hydrogen sulfide (H2S) signaling in multiple myeloma (MM) patients and to identify its effect on the proliferation of U266 cells. Methods-Bone marrow samples of 19 MM patients and 23 healthy donors were collected. qRT-PCR was performed to measure the mRNA expression levels of H2S synthases, cystathionine β synthase, and cystathionine γ lyase. ELISA assays quantified the amount of H2S produced by the two enzymes CBS and CSE. CCK-8 experiment was used to investigate the influence of the CBS inhibitor amino oxyacetic acid and the CSE inhibitor propargylglycine on the proliferation of U266 cells. Flow cytometry and western blotting were performed to determine the effects of AOAA, PAG, and NaHS on cell cycle distribution as well as Caspase-3 and Bcl-2 expression. Results-Patients with MM had higher level of CBS compared with healthy donors. AOAA significantly inhibited cell proliferation in both a time and concentration dependent characteristic, whereas PAG does not. After 24 hours of treatment, AOAA significantly elevated the G0/G1 phase proportion of cells, and reduced the cell distribution in both S and G2/M phases, while NaHS accelerated cell cycle progression by reducing the relative number of cells in G0/G1 phase and increasing the proportion of cells in the G2/M phase. Moreover, AOAA abolished the impact of NaHS on cell cycle progression of U266 cells. AOAA treatment also led to a significant decrease in Bcl-2 expression and dramatic increase in Caspase-3 expression, though NaHS reversed these effects. Conclusion-CBS/H2S system might have a certain effect on the proliferation and apoptosis of MM cells.

Discovery, Functional Characterization and Therapeutic Targeting of Lnc-17-92 Metabolic Signaling in Multiple Myeloma

Long noncoding RNAs (lncRNAs) play key roles in regulating chromatin dynamics and gene expression. We have recently described the lncRNA landscape in multiple myeloma (MM) and reported their role as independent risk predictors for clinical outcome, providing a strong rationale to evaluate their molecular and biological impact in the disease. From this lncRNA profile, we have identified lnc-17-92 as an independent risk predictor highly correlating with EFS and OS in newly-diagnosed MM providing rationale for its further evaluation in MM.

Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options

Aberrant activation of Wnt/β-catenin signaling plays a central role in the pathogenesis of a wide variety of malignancies and is typically caused by mutations in core Wnt pathway components, driving constitutive, ligand-independent signaling. In multiple myelomas (MMs), however, these pathway intrinsic mutations are rare despite the fact that most tumors display aberrant Wnt pathway activity. Recent studies indicate that this activation is caused by genetic and epigenetic lesions of Wnt regulatory components, sensitizing MM cells to autocrine Wnt ligands as well as to paracrine Wnts emanating from the bone marrow niche.

IL6 Promotes a STAT3-PRL3 Feedforward Loop via SHP2 Repression in Multiple Myeloma.

Overexpression of PRL-3, an oncogenic phosphatase, was identified as a novel cluster in patients with newly diagnosed multiple myeloma. However, the regulation and oncogenic activities of PRL-3 in multiple myeloma warrant further investigation. Here, we report that IL6 activates STAT3, which acts as a direct transcriptional regulator of PRL-3. Upregulation of PRL-3 increased myeloma cell viability and rephosphorylated STAT3 in a biphasic manner through direct interaction and deactivation of SHP2, thus blocking the gp130 (Y759)-mediated repression of STAT3 activity. Abrogation of PRL-3 reduced myeloma cell survival, clonogenicity, and tumorigenesis, and detailed mechanistic studies revealed "deactivation" of effector proteins such as Akt, Erk1/2, Src, STAT1, and STAT3. Furthermore, loss of PRL-3 efficiently abolished nuclear localization of STAT3 and reduced its occupancy on the promoter of target genes c-Myc and Mcl-1, and antiapoptotic genes Bcl2 and Bcl-xL. PRL-3 also played a role in the acquired resistance of myeloma cells to bortezomib, which could be overcome by PRL-3 silencing. Of clinical relevance, STAT3 and PRL-3 expression was positively correlated in five independent cohorts, and the STAT3 activation signature was significantly enriched in patients with high PRL-3 expression. Furthermore, PRL-3 could be used as a biomarker to identify high-risk patients with multiple myeloma that exhibited poor prognosis and inferior outcome even when treated with novel combinational therapeutics (proteasome inhibitors and immunomodulatory imide drugs). Conclusively, our results support a feedforward mechanism between STAT3 and PRL-3 that prolongs prosurvival signaling in multiple myeloma, and suggest targeting PRL-3 as a valid therapeutic opportunity in multiple myeloma. SIGNIFICANCE: IL6 promotes STAT3-dependent transcriptional upregulation of PRL-3, which in turn re-phosphorylates STAT3 and aberrantly activates STAT3 target genes, leading to bortezomib resistance in multiple myeloma.

Clinical significance of CXCR5-CXCL13 signaling in multiple myeloma.

e19517 Background: Multiple myeloma (MM) is characterized by the neoplastic proliferation of plasma cells resulting in monoclonal immunoglobulins in the blood, bone marrow, and urine (1). According to a 2019 American Cancer Society (ACS) report, MM is now classified as subtypes of non-Hodgkin lymphoma (NHL). ACS expects there will be ~32,110 new MM cases (18,130 in men and 13,980 in women) and ~12,960 (6,990 in men and 5,970 in women) anticipated deaths in 2019. MM is a heterogenous disease; characterizing its molecular phenotypes is important for effective treatment of MM and predicting relapse. CXCL13-CXCR5 interactions are involved in malignancy cell homing, adhesion, signal transduction, and calcium flux, all of which promote MM progression. Methods: We analyzed RNA-sequence data from matched normal, primary and relapsed MM cases. Patient control mRNA samples matched primary tumor and relapse samples were acquired from dbGap (database of Genotypes and Phenotypes) to evaluate the mRNA expression patterns of CXCL13, CXCR5 and associated genes in MM. A bioinformatics strategy was used to integrate published genomic data from MM patients (n = 480) and identify genes associated with CXCL13-CXCR5 signaling. DESeq analysis was used to determine differentially expressed genes between normal tissues, primary tumor and relapse groups. Weighted gene co-expression network analysis (WGCNA) identified clusters of genes significantly associated with the molecular phenotypes of MM. Results: Notably, gene expression driven by NFAT and JUN, known to be activated by the CXCL13-CXCR5 axis, and plasma cell signaling pathways significantly correlated with select MM molecular phenotypes and patient survival. Ingenuity pathway analysis (IPA) was performed to analyze upstream regulators, gene interaction networks and canonical pathways. Taken together, our data show CXCR5-CXCL13 signaling networks are significantly expressed and associated with MM pathogenesis and plasma clonality. Conclusions: This study provides a better understanding of the heterogeneous nature of MM and the role of the CXCL13-CXCR5 axis in MM.

Profiling of Oncogenic Signaling in Multiple Myeloma — Association with Biology, Disease Progression and Prognosis

Introduction: The pathogenesis of multiple myeloma (MM) involves complex genetic and epigenetic alterations, which ultimately affect oncogenic signaling networks. Analysis of pathway activation in primary MM cells within their native microenvironment will not only increase our understanding of MM cell biology but may open up new avenues towards tailored therapies that target individually activated signaling cascades.Methods: We established a (phospho-) immunohistochemistry (IHC)-assay to analyze key activation markers of oncogenic pathways and networks, namely RAS/RAF/MEK/ERK, PI3K/AKT, JAK/STAT3, canonical NFκB, and cMyc, in an exploratory cohort of 459 bone marrow (BM) biopsies from patients with smoldering MM (SMM), newly diagnosed symptomatic (NDMM) and refractory MM (RRMM). An independent validation cohort comprised BM samples from 462 NDMM patients who were uniformly treated within clinical trials. To integrate activation intensity and spread scores for each pathway and sample, non-linear principal component analysis (PCA) was applied.Results: Pathway profiling in progressing stages of plasma cell disorders showed the canonical NFκB pathway as nearly universally activated in SMM (97% of patients); signals for AKT, ERK, STAT3, and cMyc were only seen in subsets of SMM patients (≤33%), with STAT3 being the least activated pathway (6%). In NDMM and RRMM, NFκB activation significantly decreased to 90% and 82% of patients, respectively (p=0.02). In contrast, we observed a significant increase in activation of all other pathways and cMyc from SMM to NDMM and further to RRMM patients (p<0.001). All of these pathways were activated in at least 50% of RRMM. Analysis of 34 patients with available longitudinal samples confirmed a significant increase in strong activation of STAT3 and cMyc with progressing disease stages.Assessment of pathway clustering and interdependencies showed a strong negative correlation between NFκB and ERK in all disease stages. In contrast, a positive correlation was seen between cMyc and STAT3 as well as between AKT and ERK activation with increasing strength over progressing disease stages. Interestingly, when focusing on strong activation signals as determined by PCA, virtual absence of simultaneous activation of more than one pathway in SMM was observed, while pathway co-activation significantly increased in NDMM and RRMM (22% and 34%, p<0.001).Activation of STAT3 and expression of cMyc were significantly associated with higher ISS stage in NDMM of both the exploratory and the validation cohort. STAT3 pathway activation was also significantly associated with gain(1q21). In the validation cohort with available gene expression profiling (GEP) data, both STAT3 (p=0.02) and cMyc (p<0.001) activation showed a significant association with high risk disease according to GEP risk scores.Assessment of the impact of pathway activation on survival in the validation cohort showed expression of cMyc (p=0.01), activation of STAT3 (p=0.005) and cMyc-STAT3 co-activation (p=0.005) to negatively influence PFS. Expression of cMyc (p=0.02) and cMyc-STAT3 co-activation (p=0.02) were also significantly associated with inferior OS. STAT3 activation showed a negative trend towards inferior OS (p=0.08). Accounting for GEP-defined risk status, STAT3 pathway activation defined an ultra-high risk cohort. Assessment of differential gene expression suggested an autocrine positive feedback loop of IL-6 signaling and an enrichment of cell cycle associated genes in patients with STAT3 activation.Conclusions: Activation of either NFkB or ERK/AKT signaling appears to be a hallmark of MM. Later stages of MM are characterized by activation of additional signaling pathways that may affect therapy response. Importantly, cMyc expression and JAK/STAT3 pathway activation show adverse prognostic impact and significantly increase with disease progression. DisclosuresGoldschmidt:Sanofi: Consultancy, Research Funding; Adaptive Biotechnology: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; ArtTempi: Honoraria; Novartis: Honoraria, Research Funding; Mundipharma: Research Funding; Chugai: Honoraria, Research Funding; Takeda: Consultancy, Research Funding. Raab:BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria.

Syndecan-1 promotes Wnt/β-catenin signaling in multiple myeloma by presenting Wnts and R-spondins

AbstractMultiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/β-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate (HS) proteoglycan syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its HS chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible short hairpin RNA–mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS chains decorating syndecan-1 mediate aberrant Wnt pathway activation in MM. HS-deficient MM cells exhibited strongly decreased autocrine Wnt/β-catenin pathway activity and reduced Wnt pathway–dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side chains of syndecan-1 and that this binding contributes to paracrine Wnt pathway activation through the Wnt receptor Frizzled (Fzd). Furthermore, in an HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Fzd degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Costimulation with R-spondin and its binding to HS chains decorating syndecan-1 are indispensable for optimal stimulation of Wnt signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/β-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets in the treatment of MM.

Molecular signaling in multiple myeloma: association of RAS/RAF mutations and MEK/ERK pathway activation

Multiple myeloma (MM) is a plasma cell malignancy that is still considered to be incurable in most cases. A dominant mutation cluster has been identified in RAS/RAF genes, emphasizing the potential significance of RAS/RAF/MEK/ERK signaling as a therapeutic target. As yet, however, the clinical relevance of this finding is unclear as clinical responses to MEK inhibition in RAS-mutant MM have been mixed. We therefore assessed RAS/RAF mutation status and MEK/ERK pathway activation by both targeted sequencing and phospho-ERK immunohistochemistry in 180 tissue biopsies from 103 patients with newly diagnosed MM (NDMM) and 77 patients with relapsed/refractory MM (rrMM). We found a significant enrichment of RAS/BRAF mutations in rrMM compared to NDMM (P=0.011), which was mainly due to an increase of NRAS mutations (P=0.010). As expected, BRAF mutations were significantly associated with activated downstream signaling. However, only KRAS and not NRAS mutations were associated with pathway activation compared to RAS/BRAFwt (P=0.030). More specifically, only KRASG12D and BRAFV600E were consistently associated with ERK activation (P<0.001 and P=0.006, respectively). Taken together, these results suggest the need for a more specific stratification strategy consisting of both confirmation of protein-level pathway activation as well as detailed RAS/RAF mutation status to allow for a more precise and more effective application of targeted therapies, for example, with BRAF/MEK inhibitors in MM.

Aberrantly expressed LGR4 empowers Wnt signaling in multiple myeloma by hijacking osteoblast-derived R-spondins

The unrestrained growth of tumor cells is generally attributed to mutations in essential growth control genes, but tumor cells are also affected by, or even addicted to, signals from the microenvironment. As therapeutic targets, these extrinsic signals may be equally significant as mutated oncogenes. In multiple myeloma (MM), a plasma cell malignancy, most tumors display hallmarks of active Wnt signaling but lack activating Wnt-pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts emanating from the bone marrow (BM) niche. Here, we report a pivotal role for the R-spondin/leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) axis in driving aberrant Wnt/β-catenin signaling in MM. We show that LGR4 is expressed by MM plasma cells, but not by normal plasma cells or B cells. This aberrant LGR4 expression is driven by IL-6/STAT3 signaling and allows MM cells to hijack R-spondins produced by (pre)osteoblasts in the BM niche, resulting in Wnt (co)receptor stabilization and a dramatically increased sensitivity to auto- and paracrine Wnts. Our study identifies aberrant R-spondin/LGR4 signaling with consequent deregulation of Wnt (co)receptor turnover as a driver of oncogenic Wnt/β-catenin signaling in MM cells. These results advocate targeting of the LGR4/R-spondin interaction as a therapeutic strategy in MM.

Tofacitinib Reverses Growth Promoting Effects of the Bone Marrow Stromal Environment Though Inhibition of JAK1/STAT3 Signaling in Multiple Myeloma

Multiple myeloma is the second most common hematological malignancy in the U.S. and unfortunately remains incurable. Promising new therapies target the tumor microenvironment, in which bone marrow stromal cells (BMSCs), among others, support multiple myeloma (MM) cell growth, survival, and drug resistance. The cytokine IL-6 has been known to play a major role in this microenvironment, and constant stimulation by IL-6 persistently activates the JAK/STAT pathway and contributes to MM pathogenesis. Here, we found that inhibition of this pathway with tofacitinib, a pan-JAK inhibitor with primary activity against JAK1 and JAK3 and FDA-approved for rheumatoid arthritis, targets the tumor microenvironment and reverses the growth promotion supported by BMSCs in coculture with MM cells.Tofacitinib was previously identified in a screen of 2,684 compounds from the John Hopkins Drug Library for repurposing in the context of the MM bone marrow microenvironment; it demonstrated a strong capacity to reverse stroma-dependent growth promotion across three MM cell lines (MM1.S, OPM2, and L363) (Murnane et al., ASH 2015). RNAseq and mass spectrometry also showed an increase in JAK3 expression in MM1.S cells in the presence of BMSCs, suggesting a new role for JAK3 in supporting BMSC-mediated MM growth and survival. However, Western blot and dose-response experiments with a novel, selective JAK3 inhibitor, JAK3i (Smith et al., Nat Chem Biol 2016; 12: 373), revealed that tofacitinib's effects in coculture were almost exclusively mediated through inhibition of JAK1 and downstream inhibition of STAT3 in MM cells. Although JAK3 expression was increased in the presence of stroma, tofacitinib induced no change in JAK3 phosphorylation and expression nor activation of its downstream transcription factor, STAT5, suggesting that JAK3/STAT5 may be less central to stroma-supported MM growth.Ruxolitinib, a JAK1/2 inhibitor FDA approved for myeloproliferative neoplasms, was additionally identified in the screen as another JAK inhibitor with a similar pattern of stroma-mediated sensitization in MM1.S, OPM2 and L363. However, upon further validation we found that ruxolitinib had no growth inhibiting effect on MM cells in either monoculture or BMSC-coculture with the HS5 and HS27A stromal cell lines across 3 MM lines (JJN3, MM1.S, RPMI8266) and while it did exhibit anti-MM activity in a fourth cell line (U266), there was no additional sensitization in the coculture setting. The phenotypic differences induced by ruxolitinib in the initial screen and our studies may therefore be related to the different stromal or MM cell lines used in each setting, but we note that both in the screen and in our further validation, tofacitinib had a greater reversal of stroma protection than ruxolitinib.Surprisingly, Western blotting in MM1.S cells demonstrates that even a high dose of ruxolitinib (up to 1 μM) does not inhibit STAT3 phosphorylation in coculture, whereas tofacitinib potently inhibited this marker of JAK1 signaling. Potentially, therefore, ruxolitinib may not inhibit JAK1/STAT3 signaling as potently in MM cells despite having a more favorable in vitro IC50 vs. JAK1 than tofacitinib (3 nM vs. 112 nM, respectively). Alternatively, tofacitinib may have off-target effects beyond pan-JAK inhibition that inhibit MM cell growth. We are currently pursuing unbiased phosphoproteomic studies using mass spectrometry to elucidate global changes in phosphorylation in response to tofacitinib that may further underscore and identify these off-target effects.In addition, we demonstrate that a combination of tofacitinib with current MM therapies carfilzomib and melphalan leads to an additive effect that further increases MM cell death in the coculture context. Taken together, our results provide compelling data that tofacitinib may have general utility as an anti-MM therapy to specifically reverse the tumor-protective effects of the bone marrow microenvironment. Furthermore, our results suggest that tofacitinib may have more favorable anti-MM activity than ruxolitinib, which has already been explored in patients (protocol clinicaltrials.gov Identifier: NCT00639002). As tofacitinib is already FDA-approved, it is a promising therapy for repurposing that can be rapidly translated into clinical trials to improve outcomes in MM. DisclosuresTaunton:Global Blood Therapeutics: Equity Ownership; Cell Design Labs: Consultancy, Equity Ownership; Kezar Life Sciences: Equity Ownership, Research Funding; Pfizer: Research Funding; Principia Biopharma: Consultancy, Equity Ownership; Circle Pharma: Consultancy, Equity Ownership. Aftab:CytomX: Research Funding; Omniox, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Onyx Pharmaceuticals, Inc.: Research Funding; Cleave Biosciences, Inc.: Research Funding. Wiita:Omniox, LLC: Research Funding; Cleave Biosciences: Research Funding; Quadriga Biosciences: Research Funding; Onyx Pharmaceuticals: Research Funding.

Loss of CYLD expression unleashes Wnt signaling in multiple myeloma and is associated with aggressive disease.

Deletion or mutation of the gene encoding the deubiquitinating enzyme CYLD is a common genomic aberration in multiple myeloma (MM). However, the functional consequence of CYLD loss and the mechanism underlying its putative role as a tumor suppressor gene in the pathogenesis of MM has not been established. Here, we show that CYLD expression is highly variable in myeloma cell lines and primary MMs and that low CYLD expression is associated with disease progression from monoclonal gammopathy of undetermined significance to MM, and with poor overall and progression free-survival of MM patients. Functional assays revealed that CYLD represses MM cell proliferation and survival. Furthermore, CYLD acts as a negative regulator of NF-κB and Wnt/β-catenin signaling and loss of CYLD sensitizes MM cells to NF-κB-stimuli and Wnt ligands. Interestingly, in primary MMs, low CYLD expression strongly correlated with a proliferative and Wnt signaling-gene expression signature, but not with an NFκB target gene signature. Altogether, our findings identify CYLD as a negative regulator of NF-κB and Wnt/β-catenin signaling in MM and indicate that loss of CYLD enhances MM aggressiveness through Wnt pathway activation. Thus, targeting the Wnt pathway could be a promising therapeutic strategy in MM with loss of CYLD activity.

Abstract 2283: Molecular signaling in multiple myeloma: association of RAS/RAF mutation status and MAPK pathway activation in primary myeloma patient biopsies

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy. Recent studies have identified a dominant mutation cluster in RAS/BRAF genes, highlighting the MEK/ERK signaling pathway as a potentially promising therapeutic target. However, treatment of RAS/RAF mutant MM with the MEK inhibitor, trametinib, resulted in moderate response rates. There were, however, durable remissions in those patients (pts) who responded. This suggests a variable degree of dependency on MEK/ERK signaling in RAS/RAF mutant MM. To address this issue, we correlated RAS/RAF mutations with the RAF/MEK/ERK cascade activation in primary MM patient biopsies. Primary material consisted of bone marrow or soft tissue biopsies from 179 pts, including 102 pts with newly diagnosed myeloma (NDMM) and 77 pts with relapsed/refractory myeloma (rrMM). Activation of the RAF/MEK/ERK cascade was assessed using an immunohistochemical (IHC) assay for phospho-ERK. In parallel, KRAS, NRAS, HRAS and BRAF mutation status was assessed by targeted re-sequencing using the Ion Torrent NGS technology. The mutation frequencies found were KRAS (25%), NRAS (24%), and BRAF (8%) while no HRAS was detected, consistent with previous studies. Overall, RAS/RAF mutations were significantly more frequent in rrMM (p = 0.010), mainly driven by an increase in NRAS mutations (p = 0.017), proving the importance of RAS/RAF-mediated signaling in MM and indicating a potential role for NRAS mutations in drug resistance development. The top nine recurrently detected individual mutations were KRAS Q61H (n = 11), NRAS Q61R (11), NRAS Q61K (10), KRAS G12D/G12V (6 each), BRAF V600E (6), NRAS G13D (5), NRAS G13R/Q61H (4 each). When correlated with ERK activation status, KRAS but not NRAS mutations were associated with pathway activation compared to RAS/RAF-wild type (wt) (p = 0.003). However, these were not mutually exclusive, suggesting that ERK activation is dependent on the type of mutation rather than the fact that the RAS/RAF gene is mutated. Therefore, each recurrent RAS/RAF mutation was tested against all RAS/RAF-wt samples: Only KRAS G12D and BRAF V600E were consistently associated with ERK activation (p&amp;lt; 0.001 each). In summary, we found that RAS/RAF mutations are not generally associated with RAF/MEK/ERK pathway activation in MM. However, we did identify specific amino acid changes in KRAS and BRAF which are more likely to be associated with pathway activation and two mutations which were consistently associated with MEK/ERK signaling. These findings indicate that confirmation of protein-level pathway activation is needed when considering targeted therapy. In this regard, IHC is a cost-effective and reliable method to assess pathway activation and should therefore be considered in future. The clinical relevance of RAS/RAF mutations in MM should be further elucidated in prospective cohort studies. Citation Format: Jing Xu, Nicole Pfarr, Volker Endris, Elias K. Mai, Nur Hafzan Md Hanafiah, Nicola Lehners, Roland Penzel, Wilko Weichert, Anthony D. Ho, Peter Schirmacher, Hartmut Goldschmidt, Mindaugas Andrulis, Marc S. Raab. Molecular signaling in multiple myeloma: association of RAS/RAF mutation status and MAPK pathway activation in primary myeloma patient biopsies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2283.