Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes.

BACKGROUND: Activation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies. METHODS: This study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads. RESULTS: We show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival. CONCLUSIONS: Altogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.

Unraveling the Role of Peroxisome Proliferator-Activated Receptor ß/Δ (PPAR ß/Δ) in Angiogenesis Associated with Multiple Myeloma.

Growing evidence suggests a role for peroxisome proliferator-activated receptor ß/δ (PPAR ß/δ) in the angiogenesis, growth, and metastasis of solid tumors, but little is known about its role in multiple myeloma (MM). Angiogenesis in the bone marrow (BM) is characteristic of disease transition from monoclonal gammopathy of undetermined significance (MGUS) to MM. We examined the expression and function of PPAR ß/δ in endothelial cells (EC) from the BM of MGUS (MGEC) and MM (MMEC) patients and showed that PPAR ß/δ was expressed at higher levels in MMEC than in MGEC and that the overexpression depended on myeloma plasma cells. The interaction between myeloma plasma cells and MMEC promoted the release of the PPAR ß/δ ligand prostaglandin I2 (PGI2) by MMEC, leading to the activation of PPAR ß/δ. We also demonstrated that PPAR ß/δ was a strong stimulator of angiogenesis in vitro and that PPAR ß/δ inhibition by a specific antagonist greatly impaired the angiogenic functions of MMEC. These findings define PGI2-PPAR ß/δ signaling in EC as a potential target of anti-angiogenic therapy. They also sustain the use of PPAR ß/δ inhibitors in association with conventional drugs as a new therapeutic approach in MM.

Prognostic Factors for Overall Survival In Chronic Myeloid Leukemia Patients: A Multicentric Cohort Study by the Italian CML GIMEMA Network.

An observational prospective study was conducted by the CML Italian network to analyze the role of baseline patient characteristics and first line treatments on overall survival and CML-related mortality in 1206 newly diagnosed CML patients, 608 treated with imatinib (IMA) and 598 with 2nd generation tyrosine kinase inhibitors (2GTKI). IMA-treated patients were much older (median age 69 years, IQR 58-77) than the 2GTKI group (52, IQR 41-63) and had more comorbidities. Estimated 4-year overall survival of the entire cohort was 89% (95%CI 85.9-91.4). Overall, 73 patients (6.1%) died: 17 (2.8%) in the 2GTKI vs 56 (9.2%) in the IMA cohort (adjusted HR=0.50; 95% CI=0.26-0.94), but no differences were detected for CML-related mortality (10 (1.7%) vs 11 (1.8%) in the 2GTKIs vs IMA cohort (sHR=1.61; 0.52-4.96). The ELTS score was associated to CML mortality (high risk vs low, HR=9.67; 95%CI 2.94-31.74; p<0.001), while age (per year, HR=1.03; 95%CI 1.00-1.06; p=0.064), CCI (4-5 vs 2, HR=5.22; 95%CI 2.56-10.65; p<0.001), ELTS score (high risk vs low, HR=3.11; 95%CI 1.52-6.35, p=0.002) and 2GTKI vs IMA (HR=0.26; 95%CI 0.10-0.65, p=0.004) were associated to an increased risk of non-related CML mortality. The ELTS score showed a better discriminant ability than the Sokal score in all comparisons.

Halting the vicious cycle within the multiple myeloma ecosystem: blocking JAM-A on bone marrow endothelial cells restores angiogenic homeostasis and suppresses tumor progression.

Interactions of malignant multiple myeloma (MM) plasma cells (MM-cells) with the microenvironment control MM-cell growth, survival, drug-resistance and dissemination. As in MM microvascular density increases in the bone marrow (BM), we investigated whether BM MM endothelial cells (MMECs) control disease progression via the junctional adhesion molecule A (JAM-A). Membrane and cytoplasmic JAM-A levels were upregulated in MMECs in 111 newly diagnosed (NDMM) and 201 relapsed-refractory (RRMM) patients compared to monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Elevated membrane expression of JAM-A on MMECs predicted poor clinical outcome. Mechanistically, addition of recombinant JAM-A to MMECs increased angiogenesis whereas its inhibition impaired angiogenesis and MM growth in 2D and 3D in vitro cell culture and chorioallantoic membrane-assays. To corroborate these findings, we treated MM bearing mice with JAM-A blocking mAb and demonstrated impaired MM progression corresponding to decreased MM-related vascularity. These findings support JAM-A as an important mediator of MM progression through facilitating MM-associated angiogenesis. Collectively, elevated JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for patient survival in both NDMM and RRMM. Blocking JAM-A restricts angiogenesis in vitro, in embrio and in vivo and represents a suitable druggable molecule to halt neoangiogenesis and MM progression.

Bortezomib Treatment Modulates Autophagy in Multiple Myeloma.

Although the introduction of bortezomib as a therapeutic strategy has improved the overall survival of multiple myeloma (MM) patients, 15-20% of high-risk patients do not respond to bortezomib over time or become resistant to treatment. Therefore, the development of new therapeutic strategies, such as combination therapies, is urgently needed. METHODS: Given that bortezomib resistance may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation, and that an enormous amounts of misfolded protein is generated in myeloma plasma cells (PCs), we investigated the effect of the simultaneous inhibition of proteasome by bortezomib and autophagy by hydroxychloroquine (HCQ) treatment on PCs and endothelial cells (ECs) isolated from patients with monoclonal gammopathy of undetermined significance (MGUS) and MM. RESULTS: We found that bortezomib combined with HCQ induces synergistic cytotoxicity in myeloma PCs whereas this effect is lost on ECs. Levels of microtubule-associated protein light chain beta (LC3B) and p62 are differentially modulated in PCs and ECs, with effects on cell viability and proliferation. CONCLUSIONS: Our results suggest that treatment with bortezomib and HCQ should be associated with an anti-angiogenic drug to prevent the pro-angiogenic effect of bortezomib, the proliferation of a small residual tumor PC clone, and thus the relapse.

Bone marrow endothelial cells sustain a tumor-specific CD8+ T cell subset with suppressive function in myeloma patients.

Endothelial cells (EC) line the bone marrow microvasculature and are in close contact with CD8+ T cells that come and go across the permeable capillaries. Because of these intimate interactions, we investigated the capacity of EC to act as antigen-presenting cells (APC) and modulate CD8+ T cell activation and proliferation in bone marrow of patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance. We found that EC from MM patients show a phenotype of semi-professional APC given that they express low levels of the co-stimulatory molecules CD40, CD80 and CD86, and of the inducible co-stimulator ligand (ICOSL). In addition, they do not undergo the strong switch from immunoproteasome to standard proteasome subunit expression which is typical of mature professional APC such as dendritic cells. EC can trap and present antigen to CD8+ T cells, stimulating a central memory CD8+ T cell population that expresses Foxp3 and produces high amounts of IL-10 and TGF-ß. Another CD8+ T cell population is stimulated by professional APC, produces IFN-γ, and exerts antitumor activity. Thus, two distinct CD8+ T cell populations coexist in the bone marrow of MM patients: the first population is sustained by EC, expresses Foxp3, produces IL-10 and TGF-ß, and exerts pro-tumor activity by negatively regulating the second population. This study adds new insight into the role that EC play in MM biology and describes an additional immune regulatory mechanism that inhibits the development of antitumor immunity and may impair the success of cancer immunotherapy.

Inhibition of mTOR complex 2 restrains tumor angiogenesis in multiple myeloma.

The mammalian Target of Rapamycin (mTOR) is an intracellular serine/threonine kinase that mediates intracellular metabolism, cell survival and actin rearrangement. mTOR is made of two independent complexes, mTORC1 and mTORC2, activated by the scaffold proteins RAPTOR and RICTOR, respectively. The activation of mTORC1 triggers protein synthesis and autophagy inhibition, while mTORC2 activation promotes progression, survival, actin reorganization, and drug resistance through AKT hyper-phosphorylation on Ser473. Due to the mTOR pivotal role in the survival of tumor cells, we evaluated its activation in endothelial cells (ECs) from 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 47 patients with multiple myeloma (MM), and its involvement in angiogenesis. MM-ECs showed a significantly higher expression of mTOR and RICTOR than MGUS-ECs. These data were supported by the higher activation of mTORC2 downstream effectors, suggesting a major role of mTORC2 in the angiogenic switch to MM. Specific inhibition of mTOR activity through siRNA targeting RICTOR and dual mTOR inhibitor PP242 reduced the MM-ECs angiogenic functions, including cell migration, chemotaxis, adhesion, invasion, in vitro angiogenesis on Matrigel®, and cytoskeleton reorganization. In addition, PP242 treatment showed anti-angiogenic effects in vivo in the Chick Chorioallantoic Membrane (CAM) and Matrigel® plug assays. PP242 exhibited a synergistic effect with lenalidomide and bortezomib, suggesting that mTOR inhibition can enhance the anti-angiogenic effect of these drugs. Data to be shown indicate that mTORC2 is involved in MM angiogenesis, and suggest that the dual mTOR inhibitor PP242 may be useful for the anti-angiogenic management of MM patients.

Health-related quality of life and burden of fatigue in patients with primary immune thrombocytopenia by phase of disease.

The main objective of this study was to compare health-related quality of life (HRQOL) of primary immune thrombocytopenia (pITP) patients with that of general population, overall, and by patient group (i.e., newly diagnosed, persistent, and chronic patients). Fatigue was also investigated as a secondary objective. Overall, 424 adult patients were enrolled in a multicenter observational study and the control group consisted of a representative sample from the general population. Propensity score matching plus further multivariate linear regression adjustment was used to compare HRQOL outcomes between pITP patients and general population. Mean age of patients was 54 years. Of those with HRQOL assessment, 99 patients (23.6%) were newly diagnosed, 53 (12.6%) were persistent, and 268 (63.8%) were chronic pITP patients. Comparison by patient group versus their respective peers in the general population revealed greater impairments in persistent pITP patients. Persistent pITP patients reported clinically meaningful impairments in physical functioning (-15; 95% CI -24.1 to -5.8; P = 0.002), social functioning (-15.3; 95% CI -25.5 to -5.1; P = 0.004), role physical (-28.4; 95% CI -43.1 to -13.7; P < 0.001), role emotional (-23.9; 95% CI -40.1 to -7.7; P = 0.004), and mental health scales (-11.3; 95% CI -21.2 to -1.4; P = 0.026) of the SF-36 questionnaire. Higher fatigue severity was associated with lower physical and mental HRQOL outcomes. Our findings suggest that the burden of the disease and treatment might depend on the disease phase and that persistent pITP patients are the most vulnerable subgroup. Am. J. Hematol. 91:995-1001, 2016. © 2016 Wiley Periodicals, Inc.

Dendritic cells accumulate in the bone marrow of myeloma patients where they protect tumor plasma cells from CD8+ T-cell killing.

Many researchers have speculated that the clinical progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is driven by defects in dendritic cell (DC) function. However, evidence supporting this assumption is controversial, and no mechanism for the putative DC dysfunction has been demonstrated thus far. We studied DC subsets from the bone marrow of MM patients compared with those of MGUS patients and control subjects. We found that myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) accumulate in the bone marrow during the MGUS-to-MM progression. After engulfment of apoptotic tumor plasma cells via CD91, bone marrow mDCs and pDCs mature and are able to activate tumor-specific CD8(+) T cells. However, by interacting directly with CD28 on live (nonapoptotic) tumor plasma cells, bone marrow mDCs downregulate the expression of proteasome subunits in these cells, thus enabling their evasion from human leukocyte antigen (HLA) class I-restricted CD8(+) T-cell killing. These results suggest that DCs play a dual, but opposing, role in MM: for one, DCs activate CD8(+) T cells against tumor plasma cells and, for the other, DCs protect tumor plasma cells from CD8(+) T-cell killing. This information should be taken into account in designing immunotherapy approaches to enhance immune surveillance in MGUS and to break down immune tolerance in MM.

A HGF/cMET autocrine loop is operative in multiple myeloma bone marrow endothelial cells and may represent a novel therapeutic target.

PURPOSE: The aim of this study was to investigate the angiogenic role of the hepatocyte growth factor (HGF)/cMET pathway and its inhibition in bone marrow endothelial cells (EC) from patients with multiple myeloma versus from patients with monoclonal gammopathy of undetermined significance (MGUS) or benign anemia (control group). EXPERIMENTAL DESIGN: The HGF/cMET pathway was evaluated in ECs from patients with multiple myeloma (multiple myeloma ECs) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies, or on refractory phase to these drugs; in ECs from patients with MGUS (MGECs); and in those patients from the control group. The effects of a selective cMET tyrosine kinase inhibitor (SU11274) on multiple myeloma ECs' angiogenic activities were studied in vitro and in vivo. RESULTS: Multiple myeloma ECs express more HGF, cMET, and activated cMET (phospho (p)-cMET) at both RNA and protein levels versus MGECs and control ECs. Multiple myeloma ECs are able to maintain the HGF/cMET pathway activation in absence of external stimulation, whereas treatment with anti-HGF and anti-cMET neutralizing antibodies (Ab) is able to inhibit cMET activation. The cMET pathway regulates several multiple myeloma EC activities, including chemotaxis, motility, adhesion, spreading, and whole angiogenesis. Its inhibition by SU11274 impairs these activities in a statistically significant fashion when combined with bortezomib or lenalidomide, both in vitro and in vivo. CONCLUSIONS: An autocrine HGF/cMET loop sustains multiple myeloma angiogenesis and represents an appealing new target to potentiate the antiangiogenic management of patients with multiple myeloma.

HIF-1α of bone marrow endothelial cells implies relapse and drug resistance in patients with multiple myeloma and may act as a therapeutic target.

PURPOSE: To investigate the role of hypoxia-inducible factor-1α (HIF-1α) in angiogenesis and drug resistance of bone marrow endothelial cells of patients with multiple myeloma. EXPERIMENTAL DESIGN: HIF-1α mRNA and protein were evaluated in patients with multiple myeloma endothelial cells (MMEC) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies or on refractory phase to these drugs, at remission; in endothelial cells of patients with monoclonal gammapathies of undetermined significance (MGUS; MGECs), and of those with benign anemia (controls). The effects of HIF-1α inhibition by siRNA or panobinostat (an indirect HIF-1α inhibitor) on the expression of HIF-1α proangiogenic targets, on MMEC angiogenic activities in vitro and in vivo, and on overcoming MMEC resistance to bortezomib and lenalidomide were studied. The overall survival of the patients was also observed. RESULTS: Compared with the other endothelial cell types, only MMECs from 45% of relapsed/refractory patients showed a normoxic HIF-1α protein stabilization and activation that were induced by reactive oxygen species (ROS). The HIF-1α protein correlated with the expression of its proangiogenic targets. The HIF-1α inhibition by either siRNA or panobinostat impaired the MMECs angiogenesis-related functions both in vitro and in vivo and restored MMEC sensitivity to bortezomib and lenalidomide. Patients with MMECs expressing the HIF-1α protein had shorter overall survival. CONCLUSIONS: The HIF-1α protein in MMECs may induce angiogenesis and resistance to bortezomib and lenalidomide and may be a plausible target for the antiangiogenic management of patients with well-defined relapsed/refractory multiple myeloma. It may also have prognostic significance.

Pentraxin 3 (PTX3) inhibits plasma cell/stromal cell cross-talk in the bone marrow of multiple myeloma patients.

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor that binds with high affinity and selectivity to fibroblast growth factor-2 (FGF2), thus inhibiting its pro-angiogenic activity. Here we investigated the effects of PTX3 on monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patient-derived bone marrow (BM) plasma cells (PCs), endothelial cells (ECs), and fibroblasts (FBs), and assessed whether PTX3 can modulate the cross-talk between PCs and those microenvironment cells. PTX3 and FGF2 expression was evaluated by ELISA. Functional studies, including cell viability, wound healing, chemotaxis, and Matrigel(®) assays, were performed on MGUS and MM ECs and FBs upon the PTX3 treatment. Through western blot PTX3-induced modulation in FGF2/FGF receptor signalling pathways was evaluated in MGUS and MM ECs and FBs through western blot. Co-cultures between MM ECs/FBs and human PC lines were used to evaluate possible PTX3 indirect effects on MM PCs. Adhesion molecules were studied by flow cytometry. PTX3 provides a direct time- and dose-dependent apoptotic effect on MM ECs and FBs, but not on either MM primary PCs or human PC lines. PTX3 inhibits migration of MM ECs and FBs in a dose-dependent manner, and impacts in vitro and in vivo FGF2-mediated MM angiogenesis. Co-cultures of PCs and ECs/FBs show that PTX3 treatment indirectly impairs PC viability and adhesion. We conclude that PTX3 is an anti-angiogenic factor in MM and behaves as a cytotoxic molecule on MM cells by inhibiting the cross-talk between PCs and ECs/FBs.

Lenalidomide restrains motility and overangiogenic potential of bone marrow endothelial cells in patients with active multiple myeloma.

PURPOSE: To determine the in vivo and in vitro antiangiogenic power of lenalidomide, a "lead compound" of IMiD immunomodulatory drugs in bone marrow (BM) endothelial cells (EC) of patients with multiple myeloma (MM) in active phase (MMEC). EXPERIMENTAL DESIGN: The antiangiogenic effect in vivo was studied using the chorioallantoic membrane (CAM) assay. Functional studies in vitro (angiogenesis, "wound" healing and chemotaxis, cell viability, adhesion, and apoptosis) were conducted in both primary MMECs and ECs of patients with monoclonal gammopathies (MGUS) of undetermined significance (MGEC) or healthy human umbilical vein endothelial cells (HUVEC). Real-time reverse transcriptase PCR, Western blotting, and differential proteomic analysis were used to correlate morphologic and biological EC features with the lenalidomide effects at the gene and protein levels. RESULTS: Lenalidomide exerted a relevant antiangiogenic effect in vivo at 1.75 µmol/L, a dose reached in interstitial fluids of patients treated with 25 mg/d. In vitro, lenalidomide inhibited angiogenesis and migration of MMECs, but not of MGECs or control HUVECs, and had no effect on MMEC viability, apoptosis, or fibronectin- and vitronectin-mediated adhesion. Lenalidomide-treated MMECs showed changes in VEGF/VEGFR2 signaling pathway and several proteins controlling EC motility, cytoskeleton remodeling, and energy metabolism pathways. CONCLUSIONS: This study provides information on the molecular mechanisms associated with the antimigratory and antiangiogenic effects of lenalidomide in primary MMECs, thus giving new avenues for effective endothelium-targeted therapies in MM.

Bortezomib and zoledronic acid on angiogenic and vasculogenic activities of bone marrow macrophages in patients with multiple myeloma.

Bone marrow neovascularisation supports plasma cell tumour progression in patients with multiple myeloma (MM), and is partially sustained by bone marrow macrophages through their angiogenic and vasculogenic activities. As such, macrophages may be a target for antivascular treatment in MM. Here, we show that bortezomib (BZ) and zoledronic acid (ZOL) display distinct and synergistic inhibitory effects on cell proliferation, adhesion, migration and expression of angiogenic cytokines (i.e.: VEGF, bFGF, HGF and PDGF). Similar effects were found on capillarogenic organisation and expression of vascular markers in cells which became vasculogenic. VEGFR2 and ERK1/2 phosphoactivation as well as NF-kappaB activity were also inhibited. Overall these data provide evidence that the exposure of bone marrow macrophages in MM during the treatment with ZOL and BZ, alone and or in combination, impacts their angiogenic and vasculogenic properties, suggesting that these cells may be considered as a target of both drugs in MM patients.