The Role of Proteasome Inhibitors in Multiple Myeloma Bone Disease and Bone Metastasis: Effects on Osteoblasts and Osteocytes

The alterations of bone remodeling are typical of multiple myeloma (MM) patients where the uncoupled and unbalanced bone remodeling caused the onset of osteolytic lesions. Moreover, bone metastasis occurs in the majority of patients with breast and prostate cancer. Skeletal-related events negatively impact on quality of life by increasing the vulnerability to fractures. Several bone-targeting treatments have been developed to control bone pain and pathological fractures, including bisphosphonates and Denosumab. Nevertheless, these agents act by inhibiting osteoclast activity but do not improve bone formation. Proteasome inhibitors (PIs) have shown bone anabolic effects and encouraging results in stimulating osteoblast differentiation and bone healing. Among these, the first-in-class bortezomib and the second-generation PIs, carfilzomib, and ixazomib regulate the bone remodeling process by controlling the degradation of several bone proteins. PIs have been recently proven to also be efficacious in blocking MM-induced osteocyte death providing new possible therapeutic use in the management of bone loss. PIs have significant side effects that limit their use as bone anabolic strategy. Multiple alternative approaches have been made. The conjugation of PIs with bisphosphonates, which can target them to bone, showed good results in terms of bone anabolic activity. However, the clinical implications of these effects require further investigations

Role of 1q21 in Multiple Myeloma: From Pathogenesis to Possible Therapeutic Targets

Multiple myeloma (MM) is characterized by an accumulation of malignant plasma cells (PCs) in the bone marrow (BM). The amplification of 1q21 is one of the most common cytogenetic abnormalities occurring in around 40% of de novo patients and 70% of relapsed/refractory MM. Patients with this unfavorable cytogenetic abnormality are considered to be high risk with a poor response to standard therapies. The gene(s) driving amplification of the 1q21 amplicon has not been fully studied. A number of clear candidates are under investigation, and some of them (IL6R, ILF2, MCL-1, CKS1B and BCL9) have been recently proposed to be potential drivers of this region. However, much remains to be learned about the biology of the genes driving the disease progression in MM patients with 1q21 amp. Understanding the mechanisms of these genes is important for the development of effective targeted therapeutic approaches to treat these patients for whom effective therapies are currently lacking. In this paper, we review the current knowledge about the pathological features, the mechanism of 1q21 amplification, and the signal pathway of the most relevant candidate genes that have been suggested as possible therapeutic targets for the 1q21 amplicon

Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients

Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.Leukemia advance online publication, 17 July 2015; doi:10.1038/leu.2015.150

IN VITRO CHARACTERISTICS OF HEMATOPOIETIC PROGENITORS FROM PRIMARY MYELOFIBROSIS PATIENTS CORRELATE WITH IPSS/DIPSS RISK CATEGORY

Hyperplasia of morphologically abnormal megakaryocytes (MKs) is a hallmark of primary myelofibrosis (PMF) but the molecular events leading to MK abnormalities are still unclear. We previously demonstrated that, in thrombopoietin (TPO)-stimulated cultures, PMF CD34+ cells showed enhanced in vitro expansion capacity and impaired megakaryocytic differentiation compared to CD34+ cells from healthy individuals, and that the over-expression of the proto-oncogene protein kinase C epsilon (PKC) contributes to these abnormalities (Masselli et al. ASH 2013 abstr.114). Here we investigated whether clinical (included in the IPSS or DIPSS risk category) or biological (JAK2 mutational status) variables might impact the in vitro behavior of TPO-stimulated PMF CD34+ cells. We stratified 8 PMF patients according to the IPSS or DIPSS category (low/intermediate vs high risk) and JAK2 mutational status and evaluated: (1) Fold increase (FI) at day 14 of culture, (2) MK differentiation (% of CD41+ and CD42b+ cells and% of proplatelet-forming MKs) and (3) PKC protein levels (by western blot). CD34+ cells from high risk PMFs displayed increased proliferative capacity as compared to low/intermediate risk (FI: 44±0.2 vs 26.5±4.3, p=0.012), while no difference could be observed between JAK2V617F+ and JAK2V617F- PMFs (FI: 37.2±11.8 vs 27.9±5, p=0.39). Additionally, high risk PMFs revealed impaired MK differentiation potential, as indicated by the lower% of CD41+ and CD42b+ cells (respectively: 26.3±9.4 vs 54.2.6, p=0.008 and 16.1±8 vs 38.2±3, p=0.011) and proplatelet-forming MKs (0.67±0.21 vs 1.8±0.25, p=0.035). By contrast, no statistical difference was observed according to the JAK2 mutation. Finally, we found that high risk patients- derived MKs are characterized by higher expression of PKC as compared to low/intermediate risk ones (relative PKC /GAPDH OD values: 1.75±0.52 in high risk vs 0.82±0.29 in low risk, p=0.023). Conversely, PKC levels were comparable among JAK2V617F+ and JAK2V617F- PMFs (1.11±0.34 vs 1.3±0.8, p=0.72). These data indicate that the degree of in vitro growth and megakaryocytic commitment of PMF CD34+ cells is correlated to the aggressiveness of the disease (indicated by IPSS/DIPSS risk category) and not to the JAK2V617F mutation. Similarly, we found that PKC levels are significantly greater in high vs low/intermediate risk patients, leading us to speculate that PKC can be utilized as a marker of high disease burden and a more aggressive disease

The Proteasome Inhibitor Bortezomib Maintains Osteocyte Viability in Multiple Myeloma Patients by Reducing Both Apoptosis and Autophagy: A New Function for Proteasome Inhibitors

Multiple myeloma (MM) is characterized by severely imbalanced bone remodeling. In this study, we investigated the potential effect of proteasome inhibitors (PIs), a class of drugs known to stimulate bone formation, on the mechanisms involved in osteocyte death induced by MM cells. First, we performed a histological analysis of osteocyte viability on bone biopsies on a cohort of 37 MM patients with symptomatic disease. A significantly higher number of viable osteocytes was detected in patients treated with a bortezomib (BOR)-based regimen compared with those treated without BOR. Interestingly, both osteocyte autophagy and apoptosis were affected in vivo by BOR treatment. Thereafter, we checked the in vitro effect of BOR to understand the mechanisms whereby BOR maintains osteocyte viability in bone from MM patients. We found that osteocyte and preosteocyte autophagic death was triggered during coculturing with MM cells. Our evaluation was conducted by analyzing either autophagy markers microtubule-associated protein light chain 3 beta (LC3B) and SQSTM1/sequestome 1 (p62) levels, or the cell ultrastructure by transmission electron microscopy. PIs were found to increase the basal levels of LC3 expression in the osteocytes while blunting the myeloma-induced osteocyte death. PIs also reduced the autophagic death of osteocytes induced by high-dose dexamethasone (DEX) and potentiated the anabolic effect of PTH(1-34). Our data identify osteocyte autophagy as a new potential target in MM bone disease and support the use of PIs to maintain osteocyte viability and improve bone integrity in MM patients

Angiopoietin-1 and Osteopontin Expression by CD138+ Myeloma Cells Rather Than VEGF and CD45 Correlates with Bone Marrow Angiogenesis in Multiple Myeloma Patients at the Diagnosis

Bone marrow (BM) angiogenesis is increased in Multiple Myeloma (MM) patients and correlates with disease progression and patient survival. Myeloma cells secrete the main endothelial growth factor VEGF. In mouse models VEGF secretion as well as the angiogenic properties of MM cells correlate with the lack of CD45 expression by MM cells. However, recent data indicate that VEGF plasma cell expression is similar between MGUS and MM patients suggesting that other molecules could be involved. In line with this hypothesis we have recently demonstrated that myeloma cells may also produce factors with angiogenic properties as angiopoietin-1 (ANG-1) and osteopontin (OPN) that are involved in myeloma induced angiogenesis in vitro. In order to identify which factors correlate with BM angiogenesis in MM patients, we have investigated in a cohort of 121 newly diagnosed MM patients (stage I-III) the expression of the angiogenic molecules VEGF, ANG-1 and OPN and their correlation with bone marrow (BM) angiogenesis and CD45 expression by MM cells. We found that 90% of CD138+ MM cells tested were positive for VEGF mRNA. On the other hand we found that 50% and 40 % of MM patients were positive for ANG-1 and OPN mRNA respectively. Using the previously published cut off for CD45 expression we found that 61 out of 121 MM patients were positive for CD45 and 60 out of 121 were negative for CD45 expression. Any correlation was not observed between VEGF expression and BM angiogenesis in MM patients (p=0.5), whereas the number of microvessels X field was higher in Ang-1 positive patients in comparison with Ang-1 negative ones (meanSE: 6.230.2 vs. 2.940.1, median: 6.21 vs. 2.79; p=0.001,) and the microvascular density (MVD) was significantly increased (32.981.7 vs. 14.551.3, median: 34.69 vs. 13.04; p<0.01; capillaries: 26.731.3 vs. 10.420.8, median: 24.06 vs. 9.04; p<0.01, small venules: 9.56 0.5 vs. 4.140.5, median: 10.60 vs. 3.65; p<0.01). Furthermore a significantly positive correlation between Ang-1 expression and MVD was found (Pearson Chi-square: p=0.036, Cochrans Linear Trend: p=0.01). A significantly higher MVD was also observed in the group of patients positive for OPN, (meanSE: 29.10.7 vs. 17.550.37; p<0.01) and similarly, the number of microvessels per field was higher in OPN positive patients in comparison with OPN negative ones (meanSE: 6.70.15 vs. 4.280.04; p=0.05). On the other hand, any significant difference was not observed between CD45 positive and CD45 negative patients for the expression of VEGF (p=0.4), ANG-1 (p=0.3) and OPN (p=0.09). Consistently we did not find any significant difference in both MVD and number of vessels X field between CD45 positive patients as compared with CD45 negative ones (p=0.5 and p=0.4, respectively). Finally, a multivariate analysis confirmed that VEGF and CD45 did not correlate with the BM angiogenesis showing that ANG-1 expression by MM cells was more tightly correlated with MVD and the number of vessels X field as compared to OPN. Our data indicate that ANG-1 and in part OPN rather than VEGF and CD45 expression by MM cells are the critical determinants correlated with the increase of BM angiogenesis that occurs in MM patients at the diagnosis

Neurofibromatosis type I and multiple myeloma coexistence: A possible link?

The association between Neurofibromatosis type I (NF1) and multiple myeloma (MM), a plasma cell, dyscrasia is very rare. Here we put to the attention of the scientific community two new cases. The first one is a patient with active MM whereas the second with smoldering MM. Both patients present typical features of NF1 but skeletal alterations were present only in the second case including dysplasia, marked scoliosis and osteoporosis. MM osteolytic lesions were absent in both patients. In addition to the clinical diagnosis of NF1, a molecular testing for NF1 gene mutations has been performed finding that patient one was heterozygous for the c.6855C>A (Tyr2285Ter) mutation, while patient two was heterozygous for the c.7838dupC (Lys2614GlufsTer20) mutation. The two mutations were diagnosed both in genomic DNA from peripheral blood and from MM cells. The potential link between NF1 mutation and the increased risk of MM is discussed in the report