A systematic methodology for proteome-wide identification of peptides inhibiting the proliferation and migration of endothelial cells

We introduce a systematic computational methodology based on bioinformatics that has enabled us to identify and classify >120 endogenous peptide inhibitors of endothelial cell proliferation and migration. These peptides are derived from members of the type IV collagen, thrombospondin, and CXC chemokine protein families, as well as somatotropin hormones, serpins, and various kringle-containing proteins. Their activity in suppressing the proliferation and migration of endothelial cells in vitro provides proof of principle for the validity of this computational method. Interestingly, some of the peptides are derived from proteins known to be proangiogenic. By performing receptor neutralization studies, we have identified receptors to which these peptides bind. On the basis of this receptor-binding information, we evaluated several examples of peptide-based combinatorial screening strategies. In some cases, this combinatorial screening identified strong synergism between peptides. The current work provides a guideline for a computational-based peptidomics approach for the discovery of endogenous bioactive peptides.     

Single-cell atlas of the immune microenvironment reveals macrophage reprogramming and the potential dual macrophage-targeted strategy in multiple myeloma

The tumour microenvironment (TME) plays a critical role in disease progression in multiple myeloma (MM). This study aimed to present an atlas of MM-TME in disease progression and explore TME-directed therapeutic strategies. We performed single-cell RNA sequencing (scRNAseq) in samples from different disease stages. We validated the findings by bulk RNAseq, flow cytometry (FCM) and in vitro and in vivo functional experiments. We delineated a compromised TME during disease progression, characterized by enrichment of exhausted NK cells and CD8⁺ T cells and reprogramming of macrophages (MPs). The reprogrammed tumour-associated MPs (TAMs) displayed a mixed phenotype showing both M1 and M2 features, with two TAM clusters exclusively present in the MM stage showing higher M2 scores. We validated the mixed M1/M2 phenotype in TAMs in a clinical cohort and verified phagocytic dysfunction in reprogrammed TAMs. Cellular interaction analysis identified two enriched ligand–receptor pairs between MPs and malignant plasma cells (PCs), including the SIRPA-CD47 pathway suppressing phagocytosis and the CD74–MIF (macrophage inhibitory factor) reshaping the phenotype of MPs. The expression of CD47 and MIF correlated with disease progression and adverse outcomes. We designed a dual-MP-targeted strategy by combining an anti-CD47 antibody and MIF inhibitor to activate phagocytosis and repolarize MP to a functional phenotype and proved its potent antitumour effect in vitro and in vivo. We drafted alterations in MM-TME during disease progression and unravelled TAM's reprogramming. The dual MP-targeted approach blocking both CD47 and MIF showed potent antitumour effects.     

Bone marrow of patients with active multiple myeloma: Angiogenesis and plasma cell adhesion molecules LFA-1, VLA-4, LAM-1, and CD44

Bone marrow plasma cells and stromal cells in multiple myeloma (MM) have been shown to be capable of releasing cytokines with angiogenic properties. Plasma cells can also express adhesion molecules controlling their adhesive interactions with endothelial cells. In the present study, we have evaluated by immunohistochemistry the extent of angiogenesis in the bone marrow of: a) 51 patients with active and non-active MM; b) 25 patients with monoclonal gammopathy of undetermined significance (MGUS). Plasma cells were investigated by flow cytometry for the expression of the adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. The results showed that, while angiogenesis was very low or absent in patients with MGUS and non-active MM, it increased markedly in those with active MM. The highest detectability of plasma cell adhesion molecules, except LAM-1, was also found in these patients. The functional significance of these findings is unknown. Their consistent occurrence in the bone marrow of active myeloma patients, however, strongly suggests that more frequent adhesive interactions between plasma cells and their microvasculature underlie tumor dissemination.     

Plasma levels of tumour necrosis factor alpha and interleukin-6 predict progression-free survival following thalidomide therapy in patients with previously untreated multiple myeloma

We studied marrow angiogenesis and plasma levels of angiogenic cytokines in 38 patients receiving thalidomide therapy for previously untreated myeloma. The effect of therapy and the relationship of cytokine levels to myeloma cell proliferation, bone marrow microvessel density and progression-free survival (PFS) were studied. High pretreatment tumour necrosis factor-alpha (TNFalpha) levels (> 11 pg/ml) and increased interleukin (IL)-6 of > 2 pg/ml predicted for poorer PFS (TNFalpha, 48% versus 74% at 2 years, P = 0.01; IL-6, 24% versus 70% at 2 years, P = 0.01). None of the other parameters predicted response or PFS, and no significant changes in cytokine levels occurred with therapy.     

The absence of CD56 on malignant plasma cells in the cerebrospinal fluid is the hallmark of multiple myeloma involving central nervous system

By immunohistochemistry, the CD56-positive myeloma cells were detected in three (38%) bone marrow (BM) and one (13%) cerebrospinal fluid (CSF) samples from eight patients with multiple myeloma involving the central nervous system (CNS MM). Of the three patients with CD56-positive BM myeloma cells, two had CSF myeloma cells negative for CD56. In a control cohort of 84 MM patients without CNS involvement, the BM myeloma cells were CD56-positive in 68 (80%) cases (P < 0.0001). The prevalence of CD56-negative myeloma cells in the BM and CSF of our patients suggests that CD56 downregulation may play a role in the pathogenesis of CNS MM.     

Treatment response to bortezomib in multiple myeloma correlates with plasma hepatocyte growth factor concentration and bone marrow thrombospondin concentration

Multiple myeloma (MM) is associated with increased rate of bone marrow angiogenesis. Increased concentration of hepatocyte growth factor (HGF) is associated with poor prognosis in patients treated with conventional chemotherapy or thalidomide. We have shown previously that decreased level of thrombospondin, an angiogenesis inhibitor, correlates with poor response to high‐dose chemotherapy. The aim of our current study was to evaluate association between therapeutic response to bortezomib and thrombospondin and HGF levels. Peripheral blood plasma concentration of HGF and bone marrow plasma concentration of thrombospondin were measured in patients with MM prior to the initiation of bortezomib therapy. Overall, 58 patients were enrolled, 44/58 (76%) of them with relapsed disease. Treatment outcomes were analyzed for possible associations with pretreatment HGF and thrombospondin levels. Patients who achieved complete response had significantly higher pretreatment HGF levels and lower pretreatment thrombospondin levels than others. More than 70% of patients with low pretreatment HGF and high pretreatment thrombospondin concentrations achieved very good partial response or complete response, in contrast to only 20% of patients with high HGF and low thrombospondin levels. High pretreatment thrombospondin and low pretreatment HGF concentrations are associated with therapeutic response to bortezomib in patients with MM.     

The molecular spectrum and clinical impact of DIS3 mutations in multiple myeloma

Multiple myeloma (MM) is a plasma cell neoplasm that presents with a major biological and clinical heterogeneity. We here investigated the spectrum of clonal and subclonal mutations of DIS3, an active part of the exosome complex, that may play a role in the development or progression of MM. The whole coding sequence of DIS3 was subjected to deep sequencing in 81 uniformly‐treated MM patients and 12 MM cell lines and the overall occurrence of DIS3 mutations as well as the presence of DIS3 mutations in minor and major subclones were correlated with cytogenetic alterations and clinical parameters. Our study identified DIS3 mutations in 9/81 patients that were associated with 13q14 deletions and IGH translocations on the cytogenetic level. Specifically, we detected seven novel somatic DIS3 single nucleotide variants (SNVs) and defined three hot spot mutations within the RNB domain. Lastly, we found a trend towards a shorter median overall survival for patients with DIS3 mutations, and patients carrying DIS3 mutations in minor subclones of their tumours showed a significantly worse response to therapy compared to patients with DIS3 mutations in the major subclone.     

Anti-tumor activity and signaling events triggered by the isothiocyanates, sulforaphane and phenethyl isothiocyanate, in multiple myeloma

Background

Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties.

Design and Methods

We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting.

Results

We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients‘ myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G₂/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/β and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma.

Conclusions

Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.     

Prognostic value of bone marrow angiogenesis in multiple myeloma: use of light microscopy as well as computerized image analyzer in the assessment of microvessel density and total vascular area in multiple myeloma and its correlation with various clinical, histological, and laboratory parameters

We studied the prognostic value of parameters of angiogenesis on bone marrow biopsies in newly diagnosed multiple myeloma (MM) patients. Angiogenesis parameters studied were the microvessel count done manually on light microscopy (MVD-A), microvessel count done by using computerized image analyzer (MVD-B), and total vascular area (TVA) measured by computerized image analyzer. One hundred ten newly diagnosed cases of MM treated at Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, were analyzed with respect to clinical features, laboratory findings, histological features, angiogenesis parameters, and responses to the treatment on follow-up. Twenty age- and sex-matched controls were studied for comparing with angiogenesis of the test cases. Bone marrow microvessels were examined using immunohistochemical staining for CD34. MVD-A (range 4.9-85.2; mean 28.2; SD 19.4), MVD-B (range 2.0-26.9; mean 11.7; SD 5.9), and TVA measured in percentage (range 0.1-17.1; mean 2.4; SD 2.5) were measured for test cases (n = 110). Grading of angiogenesis parameters of the test cases were done; such that angiogenesis parameters of controls (taken as baseline) were grade I. There was a statistically highly significant correlation between (MVD-A vs MVD-B, pcc = 0.92; MVD-A vs TVA, pcc = 0.78; MVD-B vs TVA, pcc = 0.76). The myeloma cases had significantly higher angiogenesis parameters when compared with controls (Kruskall-Wallis test, P < 0.001). "Complete responders" (n = 38/110) had significant lower angiogenesis (Mann-Whitney U test, P < 0.001) than "nonresponders" (n = 72/110). On treatment follow-up "rapid disease progressors" had the highest levels of angiogenesis (mean rank for MVD-A = 84.7, MVD-B = 82.1, and TVA = 81.1). On multivariate (logistic regression) analysis, factors found to have independent prognostic significance in complete responders (adjusted odd ratio (95% CI, P value)] were: (a) MVD-B grade I [0.134 (0.10-0.16, P < 0.001)], (b) clinical substage A [0.163 (0.12-0.19, P = 0.008)], (c) Bartl's histological stage II & I [0.262 (0.2-0.32, P = 0.021)], (d) MVD-A grade I [0.28 (0.22-0.36, P = 0.03)], (e) beta2 microglobulin levels less than 3,400 ng/dl [0.31 (0.23-0.42, P = 0.04)]. Kaplan-Meier survival analysis for myeloma-related death (n = 16) shows a mean survival time (in months) of 24.75; SE = 3; 95% CI = 21-28. We conclude that MVD (particularly MVD-B) is a very good predictor for the complete response in patients of MM and should be done routinely on bone marrow biopsies.     

Interleukin-6 inhibits the chemotaxis of human malignant plasma cell lines

The chemotaxis of human malignant plasma cells is promoted by two extracellular matrix proteins (ECMs): fibronectin (FN) and laminin (LN). We examined the effect of the supernatant from a bone marrow stroma cell line, KM-101, on the chemotaxis of human malignant plasma cell lines to assess the chemotaxis-regulatory roles of the bone marrow microenvironment. Five human malignant plasma cell lines, FR4ds, OPM-1ds, U266/B1, RPMI-8226 and ARH-77 showed different profiles of the expression of β1 integrins of FN and LN receptors. FR4ds, OPM-1ds and U266/B1 cells showed chemotaxis promoted by FN (Ch^FN) and LN (Ch^LN). ARH-77 cells showed Ch^FN but not Ch^LN. RPMI-8226 cells did not show either Ch^FN or Ch^LN. The supernatant from KM-101 cells inhibited the chemotaxis of each of these cell lines regardless of whether the chemotaxis was promoted by FN or LN. Among the cytokines produced by KM-101 cells, it was postulated that IL-6 mediated this inhibitory effect because anti-IL-6 monoclonal antibody (MoAb) and anti-IL-6 receptor MoAb significantly reversed the inhibition. Recombinant IL-6 (rIL-6) also exhibited a similar inhibitory effect. Because anti-gp130 MoAb significantly reversed the chemotaxis inhibitory effect of rIL-6, the inhibitory signal is probably transduced via the signal transducing receptor component, gp130. The chemotaxis-regulatory effect is another previously unrecognized function of this pleiotropic cytokine, IL-6. High levels of IL-6 in the bone marrow microenvironment of patients with multiple myeloma appears to be favourable for the localization of myeloma cells there.     

The use of intravenous intermediate dose melphalan and dexamethasone as induction treatment in the management of de novo multiple myeloma

Abstract: The variable absorption of melphalan from the gastrointestinal tract results in response rates between 40 and 60%. High dose melphalan increases response rates but at the cost of increased morbidity and mortality. We have investigated intravenous intermediate dose melphalan and dexamethasone in the treatment of patients presenting with de novo multiple myeloma with the object of reducing toxicity while preserving an improved response rate compared to oral melphalan and prednisolone. The results show that this treatment can be delivered safely on an outpatient basis in patients up to the age of 78 yr; 82% of patients achieved an objective response and 30% a complete haematological and clinical remission. Median overall survival for the whole group is 37 months.     

The selective adhesion molecule inhibitor Natalizumab decreases multiple myeloma cell growth in the bone marrow microenvironment: therapeutic implications

Recent advances regarding the introduction of anti-adhesion strategies as a novel therapeutic concept in oncology hold great promise. Here we evaluated the therapeutic potential of the new-in-class-molecule selective-adhesion-molecule (SAM) inhibitor Natalizumab, a recombinant humanized IgG4 monoclonal antibody, which binds integrin-α4, in multiple myeloma (MM). Natalizumab, but not a control antibody, inhibited adhesion of MM cells to non-cellular and cellular components of the microenvironment as well as disrupted the binding of already adherent MM cells. Consequently, Natalizumab blocked both the proliferative effect of MM-bone marrow (BM) stromal cell interaction on tumour cells, and vascular endothelial growth factor (VEGF)-induced angiogenesis in the BM milieu. Moreover, Natalizumab also blocked VEGF- and insulin-like growth factor 1 (IGF-1)-induced signalling sequelae triggering MM cell migration. In agreement with our in vitro results, Natalizumab inhibited tumour growth, VEGF secretion, and angiogenesis in a human severe combined immunodeficiency murine model of human MM in the human BM microenvironment. Importantly, Natalizumab not only blocked tumour cell adhesion, but also chemosensitized MM cells to bortezomib, in an in vitro therapeutically representative human MM-stroma cell co-culture system model. Our data therefore provide the rationale for the clinical evaluation of Natalizumab, preferably in combination with novel agents (e.g. bortezomib) to enhance MM cytotoxicity and improve patient outcome.     

The importance of frailty assessment in multiple myeloma: a position statement from the Myeloma Scientific Advisory Group to Myeloma Australia

Multiple myeloma (MM) is a disease of older people, yet factors relating to comorbidity and frailty may threaten treatment tolerability for many of this heterogenous group. There has been increasing interest in defining specific and clinically relevant frailty assessment tools within the MM population, with the goal of using these frailty scores, not just as a prognostic instrument, but also as a predictive tool to allow for a frailty-adapted treatment approach. This paper reviews the various frailty assessment frameworks used in the evaluation of patients with MM, including the International Myeloma Working Group Frailty Index (IMWG-FI), the Mayo Frailty Index and the simplified frailty scale. While the IMWG-FI remains the most widely accepted tool, the simplified frailty scale is the most user-friendly in busy day-to-day clinics based on its ease of use. This paper summarises the recommendations from the Myeloma Scientific Advisory Group (MSAG) of Myeloma Australia, on the use of frailty assessment tools in clinical practice and proposes a frailty-stratified treatment algorithm to aid clinicians in tailoring therapy for this highly heterogeneous patient population.