A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

CD138 (also termed SDC1) has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug-resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down-regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138-negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

Results of a phase 2 trial of the single-agent histone deacetylase inhibitor panobinostat in patients with relapsed/refractory Waldenström macroglobulinemia.

The present study aimed to determine the safety and activity of the histone deacetylase inhibitor panobinostat in patients with relapsed/refractory Waldenström macroglobulinemia (WM). Eligibility criteria included patients with relapsed/refractory WM with any number of prior therapies. Patients received panobinostat at 30 mg 3 times a week; 12 of 36 (33%) patients were enrolled at 25 mg dose. A total of 36 patients received therapy. The median age was 62 years (range, 47-80) and the median number of prior therapies was 3 (range, 1-8). All of the patients had received prior rituximab. Minimal response (MR) or better was achieved in 47% of patients (90% confidence interval [CI], 33-62), with 22% partial remissions and 25% MR. In addition, 18 (50%) patients achieved stable disease and none showed progression while on therapy. The median time to first response was 1.8 months (range, 1.7-3.2). The median progression-free survival was 6.6 months(90% CI, 5.5-14.8). Grade 3 and 4 toxicities included thrombocytopenia (67%), neutropenia (36%), anemia (28%), leukopenia (22%), and fatigue (11%). We conclude that panobinostat is an active therapeutic agent in patients with relapsed/ refractory WM. This study (www.clinicaltrials.gov identifier: NCT00936611) establishes a role for histone deacetylase inhibitors as an active class of therapeutic agents in WM.

PI3KCA plays a major role in multiple myeloma and its inhibition with BYL719 decreases proliferation, synergizes with other therapies and overcomes stroma-induced resistance.

The phosphatidylinositide 3-kinase (PI3K) pathway is activated and correlated with drug resistance in multiple myeloma (MM). In the present study we investigated the role of PI3KCA (PI3K-α) in the progression and drug resistance in MM. We showed that the gene expression of PI3KCA isoform was higher in MM compared to normal subjects. BYL719, a novel and specific PI3KCA inhibitor inhibited the survival of primary MM cells and cell lines but not normal peripheral blood mononuclear cells. BYL719 induced the apoptosis of MM cells and inhibited their cell cycle by causing G1 arrest. BYL719 inhibited PI3K signalling, decreased proliferation and cells cycle signalling, and induced apoptosis signalling in MM cells. Finally, BYL719 synergized with bortezomib and carfilzomib, and overcame drug resistance induced by bone marrow stroma. These results were confirmed using in silico simulation of MM cell lines, BYL719 and bortezomib, and showed similar trends in survival, proliferation, apoptosis, cell signalling and synergy with drugs. In conclusion, PI3KCA plays a major role in proliferation and drug resistance of MM cells, the effects of which were inhibited with BYL719. These results provide a preclinical basis for a future clinical trial of BYL719 in MM as a single agent or in combination with other drugs.

Hypoxia promotes dissemination of multiple myeloma through acquisition of epithelial to mesenchymal transition-like features.

The spread of multiple myeloma (MM) involves (re)circulation into the peripheral blood and (re)entrance or homing of MM cells into new sites of the BM. Hypoxia in solid tumors was shown to promote metastasis through activation of proteins involved in the epithelial-mesenchymal transition (EMT) process. We hypothesized that MM-associated hypoxic conditions activate EMT-related proteins and promote metastasis of MM cells. In the present study, we have shown that hypoxia activates EMT-related machinery in MM cells, decreases the expression of E-cadherin, and, consequently, decreases the adhesion of MM cells to the BM and enhances egress of MM cells to the circulation. In parallel, hypoxia increased the expression of CXCR4, consequently increasing the migration and homing of circulating MM cells to new BM niches. Further studies to manipulate hypoxia to regulate tumor dissemination as a therapeutic strategy are warranted.

P-selectin glycoprotein ligand regulates the interaction of multiple myeloma cells with the bone marrow microenvironment.

Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in the pathogenesis of MM and in the development of drug resistance by MM cells. Selectins are involved in extravasation and homing of leukocytes to target organs. In the present study, we focused on adhesion dynamics that involve P-selectin glycoprotein ligand-1 (PSGL-1) on MM cells and its interaction with selectins in the BM microenvironment. We show that PSGL-1 is highly expressed on MM cells and regulates the adhesion and homing of MM cells to cells in the BM microenvironment in vitro and in vivo. This interaction involves both endothelial cells and BM stromal cells. Using loss-of-function studies and the small-molecule pan-selectin inhibitor GMI-1070, we show that PSGL-1 regulates the activation of integrins and downstream signaling. We also document that this interaction regulates MM-cell proliferation in coculture with BM microenvironmental cells and the development of drug resistance. Furthermore, inhibiting this interaction with GMI-1070 enhances the sensitization of MM cells to bortezomib in vitro and in vivo. These data highlight the critical contribution of PSGL-1 to the regulation of growth, dissemination, and drug resistance in MM in the context of the BM microenvironment.

LNA-mediated anti-miR-155 silencing in low-grade B-cell lymphomas.

miR-155 acts as an oncogenic miR in B-cell lymphoproliferative disorders, including Waldenstrom macroglobulinemia (WM) and chronic lymphocytic leukemia, and is therefore a potential target for therapeutic intervention. However, efficient targeting of miRs in tumor cells in vivo remains a significant challenge for the development of miR-155-based therapeutics for the treatment of B-cell malignancies. In the present study, we show that an 8-mer locked nucleic acid anti-miR-155 oligonucleotide targeting the seed region of miR-155 inhibits WM and chronic lymphocytic leukemia cell proliferation in vitro. Moreover, anti-miR-155 delivered systemically showed uptake in the BM CD19(+) cells of WM-engrafted mice, resulting in the up-regulation of several miR-155 target mRNAs in these cells, and decreased tumor growth significantly in vivo. We also found miR-155 levels to be elevated in stromal cells from WM patients compared with control samples. Interestingly, stromal cells from miR-155-knockout mice led to significant inhibition of WM tumor growth, indicating that miR-155 may also contribute to WM proliferation through BM microenvironmental cells. The results of the present study highlight the therapeutic potential of anti-miR-155-mediated inhibition of miR-155 in the treatment of WM.

Targeting survival and cell trafficking in multiple myeloma and Waldenstrom macroglobulinemia using pan-class I PI3K inhibitor, buparlisib.

The phosphatidylinositol-3 kinase (PI3K) pathway is activated in multiple myeloma (MM) and Waldenstrom Macroglobulenima (WM), and plays a crucial role in tumor progression and drug resistance. In this study, we characterized the role of pan-class I PI3K inhibition on cell trafficking and survival of MM and WM cells. We tested the effect of pan-class I PI3K inhibition by siRNA silencing or pharmacologic inhibition with buparlisib on MM cell survival, apoptosis and cell cycle in vitro and tumor growth and mobilization of MM cells in vivo. We then evaluated buparlisib-dependent mechanisms of induced MM cell mobilization. Moreover, the effect of buparlisib on cell survival, apoptosis, and adhesion of WM cells to bone marrow stromal cells (BMSCs) has been evaluated. We showed that buparlisib induced toxicity in MM cells, supported by induction of apoptosis and cell cycle arrest. Buparlisib was also found to reduce tumor progression in vivo. Importantly, buparlisib enhanced MM cell mobilization in vivo which was driven by decreased adhesion of MM cells to BMSCs and increased chemotaxis via up-regulation of CXCR4 expression. Similar to its effects on MM cells, buparlisib also induced cell survival and apoptosis, and decreased adhesion in WM cells. These data highlight the critical contribution of class I PI3K signaling to the regulation of survival and cell dissemination in B-cell malignancies.

Defining the role of TORC1/2 in multiple myeloma.

Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment to regulate multiple cellular processes. Rapamycin and its analogs have not shown significant activity in multiple myeloma (MM), likely because of the lack of inhibition of TORC2. In the present study, we investigated the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. TORC1/2 knock-down led to significant inhibition of the proliferation of MM cells, even in the presence of BM stromal cells. We also tested INK128, a dual TORC1/2 inhibitor, as a new therapeutic agent against these MM cell lines. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin), even in the presence of cytokines or stromal cells. In vitro and in vivo studies showed that p-4EBP1 and p-Akt inhibition could be predictive markers of TORC2 inhibition in MM cell lines. Dual TORC1/2 inhibition showed better inhibition of adhesion to BM microenvironmental cells and inhibition of homing in vivo. These studies form the basis for further clinical testing of TORC1/2 inhibitors in MM.

microRNA-dependent modulation of histone acetylation in Waldenstrom macroglobulinemia.

Waldenström macroglobulinemia (WM) cells present with increased expression of microRNA-206 (miRNA-206) and reduced expression of miRNA-9*. Predicted miRNA-206- and -9*-targeted genes include histone deacetylases (HDACs) and histone acetyl transferases (HATs), indicating that these miRNAs may play a role in regulating histone acetylation. We were able to demonstrate that primary WM cells are characterized by unbalanced expression of HDACs and HATs, responsible for decreased acetylated histone-H3 and -H4, and increased HDAC activity. We next examined whether miRNA-206 and -9* modulate the aberrant expression of HDAC and HATs in WM cells leading to increased transcriptional activity. We found that restoring miRNA-9* levels induced toxicity in WM cells, supported by down-modulation of HDAC4 and HDAC5 and up-regulation of acetyl-histone-H3 and -H4. These, together with inhibited HDAC activity, led to induction of apoptosis and autophagy in WM cells. To further confirm that miRNA-9*-dependent modulation of histone acetylation is responsible for induction of WM cytotoxicity, a novel class of HDAC inhibitor (LBH589) was used; we confirmed that inhibition of HDAC activity leads to toxicity in this disease. These findings confirm that histone-modifying genes and HDAC activity are deregulated in WM cells, partially driven by the aberrant expression of miRNA-206 and -9* in the tumor clone.

Selective inhibition of chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in Waldenstrom macroglobulinemia.

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor kappaB (NF-kappaB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)-derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.

Dual targeting of the PI3K/Akt/mTOR pathway as an antitumor strategy in Waldenstrom macroglobulinemia.

We have previously shown clinical activity of a mammalian target of rapamycin (mTOR) complex 1 inhibitor in Waldenstrom macroglobulinemia (WM). However, 50% of patients did not respond to therapy. We therefore examined mechanisms of activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR in WM, and mechanisms of overcoming resistance to therapy. We first demonstrated that primary WM cells show constitutive activation of the PI3K/Akt pathway, supported by decreased expression of phosphate and tensin homolog tumor suppressor gene (PTEN) at the gene and protein levels, together with constitutive activation of Akt and mTOR. We illustrated that dual targeting of the PI3K/mTOR pathway by the novel inhibitor NVP-BEZ235 showed higher cytotoxicity on WM cells compared with inhibition of the PI3K or mTOR pathways alone. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the Forkhead box transcription factors. In addition, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment, leading to significant inhibition of migration, adhesion in vitro, and homing in vivo. These studies therefore show that dual targeting of the PI3K/mTOR pathway is a better modality of targeted therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade, such as WM.

microRNA expression in the biology, prognosis, and therapy of Waldenström macroglobulinemia.

Multilevel genetic characterization of Waldenström macroglobulinemia (WM) is required to improve our understanding of the underlying molecular changes that lead to the initiation and progression of this disease. We performed microRNA-expression profiling of bone marrow-derived CD19(+) WM cells, compared with their normal cellular counterparts and validated data by quantitative reverse-transcription-polymerase chain reaction (qRT-PCR). We identified a WM-specific microRNA signature characterized by increased expression of microRNA-363*/-206/-494/-155/-184/-542-3p, and decreased expression of microRNA-9* (ANOVA; P < .01). We found that microRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-kappaB pathways. Potential microRNA-155 target genes were identified using gene-expression profiling and included genes involved in cell-cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM. We further demonstrated that therapeutic agents commonly used in WM alter the levels of the major miRNAs identified, by inducing downmodulation of 5 increased miRNAs and up-modulation of patient-downexpressed miRNA-9*. These data indicate that microRNAs play a pivotal role in the biology of WM; represent important prognostic marker; and provide the basis for the development of new microRNA-based targeted therapies in WM.

RhoA and Rac1 GTPases play major and differential roles in stromal cell-derived factor-1-induced cell adhesion and chemotaxis in multiple myeloma.

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) milieu plays a crucial role in MM pathogenesis. Stromal cell-derived factor-1 (SDF1) regulates homing of MM cells to the BM. In this study, we examined the role of RhoA and Rac1 GTPases in SDF1-induced adhesion and chemotaxis of MM. We found that both RhoA and Rac1 play key roles in SDF1-induced adhesion of MM cells to BM stromal cells, whereas RhoA was involved in chemotaxis and motility. Furthermore, both ROCK and Rac1 inhibitors reduced SDF1-induced polymerization of actin and activation of LIMK, SRC, FAK, and cofilin. Moreover, RhoA and Rac1 reduced homing of MM cells to BM niches. In conclusion, we characterized the role of RhoA and Rac1 GTPases in SDF1-induced adhesion, chemotaxis, and homing of MM cells to the BM, providing the framework for targeting RhoA and Rac1 GTPases as novel MM therapy.

MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma.

Detailed genomic studies have shown that cytogenetic abnormalities contribute to multiple myeloma (MM) pathogenesis and disease progression. Nevertheless, little is known about the characteristics of MM at the epigenetic level and specifically how microRNAs regulate MM progression in the context of the bone marrow milieu. Therefore, we performed microRNA expression profiling of bone marrow derived CD138(+) MM cells versus their normal cellular counterparts and validated data by qRT-PCR. We identified a MM-specific microRNA signature characterized by down-expression of microRNA-15a/-16 and overexpression of microRNA-222/-221/-382/-181a/-181b (P < .01). We investigated the functional role of microRNA-15a and -16 and showed that they regulate proliferation and growth of MM cells in vitro and in vivo by inhibiting AKT serine/threonine-protein-kinase (AKT3), ribosomal-protein-S6, MAP-kinases, and NF-kappaB-activator MAP3KIP3. Moreover, miRNA-15a and -16 exerted their anti-MM activity even in the context of the bone marrow milieu in vitro and in vivo. These data indicate that microRNAs play a pivotal role in the biology of MM and represent important targets for novel therapies in MM.

CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy.

The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) provides a protective environment and resistance to therapeutic agents. We hypothesized that disruption of the interaction of MM cells with their BM milieu would lead to their sensitization to therapeutic agents such as bortezomib, melphalan, doxorubicin, and dexamethasone. We report that the CXCR4 inhibitor AMD3100 induces disruption of the interaction of MM cells with the BM reflected by mobilization of MM cells into the circulation in vivo, with kinetics that differed from that of hematopoietic stem cells. AMD3100 enhanced sensitivity of MM cell to multiple therapeutic agents in vitro by disrupting adhesion of MM cells to bone marrow stromal cells (BMSCs). Moreover, AMD3100 increased mobilization of MM cells to the circulation in vivo, increased the ratio of apoptotic circulating MM cells, and enhanced the tumor reduction induced by bortezomib. Mechanistically, AMD3100 significantly inhibited Akt phosphorylation and enhanced poly(ADP-ribose) polymerase (PARP) cleavage as a result of bortezomib, in the presence of BMSCs in coculture. These experiments provide a proof of concept for the use of agents that disrupt interaction with the microenvironment for enhancement of efficacy of cytotoxic agents in cancer therapy.

A pilot study of 3D tissue-engineered bone marrow culture as a tool to predict patient response to therapy in multiple myeloma.

Cancer patients undergo detrimental toxicities and ineffective treatments especially in the relapsed setting, due to failed treatment attempts. The development of a tool that predicts the clinical response of individual patients to therapy is greatly desired. We have developed a novel patient-derived 3D tissue engineered bone marrow (3DTEBM) technology that closely recapitulate the pathophysiological conditions in the bone marrow and allows ex vivo proliferation of tumor cells of hematologic malignancies. In this study, we used the 3DTEBM to predict the clinical response of individual multiple myeloma (MM) patients to different therapeutic regimens. We found that while no correlation was observed between in vitro efficacy in classic 2D culture systems of drugs used for MM with their clinical efficacious concentration, the efficacious concentration in the 3DTEBM were directly correlated. Furthermore, the 3DTEBM model retrospectively predicted the clinical response to different treatment regimens in 89% of the MM patient cohort. These results demonstrated that the 3DTEBM is a feasible platform which can predict MM clinical responses with high accuracy and within a clinically actionable time frame. Utilization of this technology to predict drug efficacy and the likelihood of treatment failure could significantly improve patient care and treatment in many ways, particularly in the relapsed and refractory setting. Future studies are needed to validate the 3DTEBM model as a tool for predicting clinical efficacy.

Dual targeting of the proteasome regulates survival and homing in Waldenstrom macroglobulinemia.

Waldenström macroglobulinemia (WM) is an incurable low-grade B-cell lymphoma characterized by high protein turnover. We dissected the biologic role of the proteasome in WM using 2 proteasome inhibitors, NPI-0052 and bortezomib. We found that NPI-0052 inhibited proliferation and induced apoptosis in WM cells, and that the combination of NPI-0052 and bortezomib induced synergistic cytotoxicity in WM cells, leading to inhibition of nuclear translocation of p65NF-kappaB and synergistic induction of caspases-3, -8, and -9 and PARP cleavage. These 2 agents inhibited the canonical and noncanonical NF-kappaB pathways and acted synergistically through their differential effect on Akt activity and on chymotrypsin-like, caspaselike, and trypsinlike activities of the proteasome. We demonstrated that NPI-0052-induced cytotoxicity was completely abrogated in an Akt knockdown cell line, indicating that its major activity is mediated through the Akt pathway. Moreover, we demonstrated that NPI-0052 and bortezomib inhibited migration and adhesion in vitro and homing of WM cells in vivo, and overcame resistance induced by mesenchymal cells or by the addition of interleukin-6 in a coculture in vitro system. Theses studies enhance our understanding of the biologic role of the proteasome pathway in WM, and provide the preclinical basis for clinical trials of combinations of proteasome inhibitors in WM.

SDF-1/CXCR4 and VLA-4 interaction regulates homing in Waldenstrom macroglobulinemia.

Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow at the time of diagnosis, implying continuous homing of WM cells into the marrow. The mechanisms by which trafficking of the malignant cells into the bone marrow has not been previously elucidated. In this study, we show that WM cells express high levels of chemokine and adhesion receptors, including CXCR4 and VLA-4. We showed that CXCR4 was essential for the migration and trans-endothelial migration of WM cells under static and dynamic shear flow conditions, with significant inhibition of migration using CXCR4 knockdown or the CXCR4 inhibitor AMD3100. Similarly, CXCR4 or VLA-4 inhibition led to significant inhibition of adhesion to fibronectin, stromal cells, and endothelial cells. Decreased adhesion of WM cells to stromal cells by AMD3100 led to increased sensitivity of these cells to cytotoxicity by bortezomib. To further investigate the mechanisms of CXCR4-dependent adhesion, we showed that CXCR4 and VLA-4 directly interact in response to SDF-1, we further investigated downstream signaling pathways regulating migration and adhesion in WM. Together, these studies demonstrate that the CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.

Targeting NF-kappaB in Waldenstrom macroglobulinemia.

The nuclear factor-kappaB (NF-kappaB) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-kappaB pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-kappaB activity. We demonstrated that perifosine and bortezomib both targeted NF-kappaB through its recruitment to the promoter of its target gene IkappaB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-kappaB pathway.