A liquid biopsy to detect multidrug resistance and disease burden in multiple myeloma.
ABSTRACT: Multiple myeloma is an incurable cancer of bone marrow plasma cells, with a 5-year survival rate of 43%. Its incidence has increased by 126% since 1990. Treatment typically involves high-dose combination chemotherapy, but therapeutic response and patient survival are unpredictable and highly variable-attributed largely to the development of multidrug resistance (MDR). MDR is the simultaneous cross-resistance to a range of unrelated chemotherapeutic agents and is associated with poor prognosis and survival. Currently, no clinical procedures allow for a direct, continuous monitoring of MDR. We identified circulating large extracellular vesicles (specifically microparticles (MPs)) that can be used to monitor disease burden, disease progression and development of MDR in myeloma. These MPs differ phenotypically in the expression of four protein biomarkers: a plasma-cell marker (CD138), the MDR protein, P-glycoprotein (P-gp), the stem-cell marker (CD34); and phosphatidylserine (PS), an MP marker and mediator of cancer spread. Elevated levels of P-gp+ and PS+ MPs correlate with disease progression and treatment unresponsiveness. Furthermore, P-gp, PS and CD34 are predominantly expressed in CD138- MPs in advanced disease. In particular, a dual-positive (CD138-P-gp+CD34+) population is elevated in aggressive/unresponsive disease. Our test provides a personalised liquid biopsy with potential to address the unmet clinical need of monitoring MDR and treatment failure in myeloma.
Project description:BACKGROUND: There is growing evidence that CD138(-) CD34(-) cells may actually be tumor stem cells responsible for initiation and relapse of multiple myeloma. However, effective drugs targeted at CD138(-) CD34(-) tumor stem cells are yet to be developed. The purpose of this study was to investigate the inhibitory effect of paclitaxel-loaded Fe3O4 nanoparticles (PTX-NPs) on CD138(-) CD34(-) tumor stem cells in multiple myeloma-bearing mice. METHODS: CD138(-) CD34(-) cells were isolated from a human U266 multiple myeloma cell line using an immune magnetic bead sorting method and then subcutaneously injected into mice with nonobese diabetic/severe combined immunodeficiency to develop a multiple myeloma-bearing mouse model. The mice were treated with Fe3O4 nanoparticles 2 mg/kg, paclitaxel 4.8 mg/kg, and PTX-NPs 0.64 mg/kg for 2 weeks. Tumor growth, pathological changes, serum and urinary interleukin-6 levels, and molecular expression of caspase-3, caspase-8, and caspase-9 were evaluated. RESULTS: CD138(-) CD34(-) cells were found to have tumor stem cell characteristics. All the mice developed tumors in 40 days after injection of 1 × 10(6) CD138(-) CD34(-) tumor stem cells. Tumor growth in mice treated with PTX-NPs was significantly inhibited compared with the controls (P < 0.005), and the groups that received nanoparticles alone (P < 0.005) or paclitaxel alone (P < 0.05). In addition, the PTX-NPs markedly inhibited interleukin-6 secretion, increased caspase-8, caspase-9, and caspase-3 expression, and induced apoptosis of tumor cells in the treated mice. CONCLUSION: PTX-NPs proved to be a potent anticancer treatment strategy that may contribute to targeted therapy for multiple myeloma tumor stem cells in future clinical trials.
Project description:The canonical plasma cell marker CD138 (syndecan-1) is highly expressed on the myeloma cell surface, but its functional role in vivo is unclear, as well as the ontogeny of CD138-high and CD138-negative (neg) myeloma cells. In this study we used an in vivo murine Vk*MYC myeloma model where CD138 is heterogeneously expressed depending on tumor size. We find that in comparison to CD138-neg myeloma cells, the CD138-high subset of myeloma cells is highly proliferative, less apoptotic, and enhanced IL-6R signaling, which is known to promote survival. In addition CD138-high myeloma engrafts better than its CD138-neg counterpart. In contrast, CD138-neg cells are more motile both in vitro and in vivo, and more readily disseminate and spread to other bones in vivo than CD138-high subset. Neutralizing CD138 rapidly triggers migration of myeloma cells in vivo and leads to intravasation, which results in increased dissemination to other bones. Both murine and human myeloma cells can rapidly recycle CD138 surface expression through endocytic trafficking, in response to serum levels. Blocking CD138 enhances myeloma sensitivity to bortezomib chemotherapy and significantly reduces tumor size compared to bortezomib treatment alone. Thus, our data show that CD138 surface expression dynamically regulates a switch between growth vs. dissemination for myeloma, in response to nutrient conditions.
Project description:Multiple myeloma (MM) is a plasma cell malignancy characterized by the presence of multiple foci in the skeleton. These distinct tumor foci indicate cycles of tumor growth and dissemination that seed new clusters and drives disease progression. Utilizing an intra-tibial Vk*MYC murine myeloma, we found that CD169 + radiation-resistant, tissue-resident macrophages (MPs) were critical for myeloma early dissemination and disease progression. While depletion of these MPs had no effect on tumor proliferation, it reduced myeloma BM egress and spreading to other bones, which improved overall survival as a single therapy and in combination with BM transplantation. Myeloma dissemination correlated with an increased inflammatory signature in BM MPs, as well as production of IL-6 and TNFα by tumor-associated MPs (TAMs). Exogenous i.v. IL-6 and TNFa could trigger myeloma intravasation in the BM by increasing vascular leakiness in the BM, and by enhancing myeloma motility by reducing CD138 adhesion. Moreover, mice lacking IL-6 had defects in myeloma dissemination as in MP-depleted recipients. While in TNFa or TNFaR deficient mice had defects in MM dissemination, engraftment was also impaired. These effects on myeloma dissemination required production of cytokines in the radiation-resistant compartment, containing these radiation-resistant BM MPs. Taken together, we propose BM egress of myeloma cells is regulated by localized inflammation in foci, driven in part by CD169 + MPs.
Project description:<h4>Background</h4>High-dose chemotherapy with autologous stem cell transplantation is a cornerstone in the first-line treatment of multiple myeloma patients. However, only few factors have been identified affecting the outcome in such patients. We hypothesised that varying levels of mobilised CD34+ cells confer prognostic information in myeloma patients undergoing high-dose chemotherapy.<h4>Methods</h4>We determined circulating CD34+ cells at the day of peripheral stem cell collection in 158 consecutive myeloma patients between January 2001 and August 2010. Patients were stratified into two groups (super vs normal mobilisers) with a cutoff of 100,000 peripheral CD34+ cells per ml.<h4>Results</h4>We found that patients with more than 100,000 peripheral CD34+ cells per ml had a better overall survival (P=0.005) and a prolonged time to progression (P=0.0398) than patients with CD34+ cell counts below 100,000 CD34+ cells per ml. High levels of CD34+ cells were an independent marker for better overall survival and time to progression in a multivariate analysis that included disease stage, response at transplant, light-chain subtype, age, sex, and height.<h4>Conclusion</h4>Our results suggest that high levels of mobilised peripheral CD34+ cells are associated with favourable outcome in myeloma patients undergoing autologous transplantation.
Project description:In myeloma, B cells and plasma cells show a clonal relationship. Clonotypic B cells may represent a tumor-initiating compartment or cancer stem cell responsible for minimal residual disease in myeloma.We report a study of 58 patients with myeloma at time of diagnosis or relapse. B cells in bone marrow were evaluated by multicolor flow cytometry and sorting. Clonality was determined by light chain and/or immunoglobulin chain gene rearrangement PCR. We also determined aldehyde dehydrogenase activity and colony formation growth. Drug sensitivity was tested with conventional and novel agents.Marrow CD19+ cells express a light chain identical to plasma cells and are therefore termed light chain restricted (LCR). The LCR B-cell mass is small in both newly diagnosed and relapsed patients (? 1%). Few marrow LCR B cells (~10%) are CD19+/CD34+, with the rest being more differentiated CD19+/CD34- B cells. Marrow LCR CD19+ B cells exhibit enhanced aldehyde dehydrogenase activity versus healthy controls. Both CD19+/CD34+ and CD19+/CD34- cells showed colony formation activity, with colony growth efficiency optimized when stroma-conditioned medium was used. B-cell progenitors showed resistance to melphalan, lenalidomide, and bortezomib. Panobinostat, a histone deacetylase inhibitor, induced apoptosis of LCR B cells and CD138+ cells. LCR B cells are CD117, survivin, and Notch positive.We propose that antigen-independent B-cell differentiation stages are involved in disease origination and progression in myeloma. Furthermore, investigations of myeloma putative stem cell progenitors may lead to novel treatments to eradicate the potential reservoir of minimal residual disease.
Project description:Interactions between the inhibitor of apoptosis protein antagonist LCL161 and the histone deacetylase inhibitor panobinostat (LBH589) were examined in human multiple myeloma (MM) cells. LCL161 and panobinostat interacted synergistically to induce apoptosis in diverse MM cell lines, including those resistant to bortezomib (PS-R). Similar interactions were observed with other histone deacetylase inhibitors (MS-275) or inhibitors of apoptosis protein antagonists (birinapant). These events were associated with downregulation of the noncanonical (but not the canonical) NF-κB pathway and activation of the extrinsic, caspase-8-related apoptotic cascade. Coexposure of MM cells to LCL161/LBH589 induced TRAF3 upregulation and led to TRAF2 and NIK downregulation, diminished expression of BCL-XL, and induction of γH2A.X. Ectopic expression of TRAF2, NIK, or BCL-XL, or short hairpin RNA TRAF3 knock-down, significantly reduced LCL161/LBH589 lethality, as did ectopic expression of dominant-negative FADD. Stromal/microenvironmental factors failed to diminish LCL161/LBH589-induced cell death. The LCL161/LBH589 regimen significantly increased cell killing in primary CD138+ cells (N = 31) and was particularly effective in diminishing the primitive progenitor cell-enriched CD138-/19+/20+/27+ population (N = 23) but was nontoxic to normal CD34+ cells. Finally, combined LCL161/LBH589 treatment significantly increased survival compared with single-agent treatment in an immunocompetent 5TGM1 murine MM model. Together, these findings argue that LCL161 interacts synergistically with LBH589 in MM cells through a process involving inactivation of the noncanonical NF-κB pathway and activation of the extrinsic apoptotic pathway, upregulation of TRAF3, and downregulation of TRAF2/BCL-XL. Notably, this regimen overcomes various forms of resistance, is active against primary MM cells, and displays significant in vivo activity. This strategy warrants further consideration in MM.
Project description:After unprecedented successes in B-cell malignancies, chimeric antigen receptor T cells have recently been investigated for the treatment of multiple myeloma. Chimeric antigen receptor targeting T cells B-cell maturation antigen (BCMA) on malignant plasma cells have led to impressive clinical responses in recent trials. However, BCMA-negative relapses have been observed, supporting the need for complementary treatment strategies. Here, we explored the feasibility of targeting CD138 (syndecan-1), a surface marker expressed on both normal and malignant plasma cells. We showed that T cells from both healthy donors and from multiple myeloma patients, when transduced with a CD138-specific chimeric antigen receptor, can eliminate tumor cell lines and primary myeloma cells both in vitro and in vivo. CD138 is also expressed by putative myeloma stem cells identified by Hoechst staining, and these cells can be eliminated by CD138-specific chimeric antigen receptor T cells. Preclinical analyses did not identify any on target off tumor cytotoxicity against normal epithelial or endothelial cells, further supporting the rationale for the use of adoptively transferred CD138-specific chimeric antigen receptor T cells for the treatment of patients with relapsed/refractory multiple myeloma.
Project description:BACKGROUND:Mechanisms by which Smac mimetics (SMs) interact with proteasome inhibitors (e.g., bortezomib) are largely unknown, particularly in multiple myeloma (MM), a disease in which bortezomib represents a mainstay of therapy. METHODS:Interactions between the clinically relevant IAP (inhibitor of apoptosis protein) antagonist birinapant (TL32711) and the proteasome inhibitor bortezomib were investigated in multiple myeloma (MM) cell lines and primary cells, as well as in vivo models. Induction of apoptosis and changes in gene and protein expression were monitored using MM cell lines and confirmed in primary MM cell populations. Genetically modified cells (e.g., exhibiting shRNA knockdown or ectopic expression) were employed to evaluate the functional significance of birinapant/bortezomib-induced changes in protein levels. A MM xenograft model was used to evaluate the in vivo activity of the birinapant/bortezomib regimen. RESULTS:Birinapant and bortezomib synergistically induced apoptosis in diverse cell lines, including bortezomib-resistant cells (PS-R). The regimen robustly downregulated cIAP1/2 but not the canonical NF-?B pathway, reflected by p65 phosphorylation and nuclear accumulation. In contrast, the bortezomib/birinapant regimen upregulated TRAF3, downregulated TRAF2, and diminished p52 processing and BCL-XL expression, consistent with disruption of the non-canonical NF-?B pathway. TRAF3 knockdown, ectopic TRAF2, or BCL-XL expression significantly diminished birinapant/bortezomib toxicity. The regimen sharply increased extrinsic apoptotic pathway activation, and cells expressing dominant-negative FADD or caspase-8 displayed markedly reduced birinapant/bortezomib sensitivity. Primary CD138+ (n?=?43) and primitive MM populations (CD138-/19+/20+/27+; n?=?31) but not normal CD34+ cells exhibited significantly enhanced toxicity with combined treatment (P?<?0.0001). The regimen was also fully active in the presence of HS-5 stromal cells or growth factors (e.g., IL-6 and VEGF). Finally, the regimen was well tolerated and significantly increased survival (P?<?0.05 and P?<?0.001) compared to single agents in a MM xenograft model. Combined treatment also downregulated cIAP1/2 and p52 while increasing PARP cleavage in MM cells in vivo. CONCLUSIONS:Our data suggest that birinapant and bortezomib interact synergistically in MM cells, including those resistant to bortezomib, through inactivation of the non-canonical NF-?B and activation of the extrinsic apoptotic pathway both in vitro and in vivo. They also argue that a strategy combining cIAP antagonists and proteasome inhibitors warrants attention in MM.
Project description:Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased ?H2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.
Project description:<h4>Purpose</h4>Multiple myeloma is a hematologic malignancy originating from clonal plasma cells. Despite effective therapies, outcomes are highly variable suggesting marked disease heterogeneity. The role of functional imaging for therapeutic management of myeloma, such as positron emission tomography with 2-deoxy-2-[¹?F]fluoro-D-glucose (¹?F-FDG-PET), remains to be determined. Although some studies already suggested a prognostic value of ¹?F-FDG-PET, more specific tracers addressing hallmarks of myeloma biology, e.g. paraprotein biosynthesis, are needed. This study evaluated the amino acid tracers L-methyl-[¹¹C]-methionine (¹¹C-MET) and [¹?F]-fluoroethyl-L-tyrosine ((¹?F-Fet) for their potential to image myeloma and to characterize tumor heterogeneity.<h4>Experimental design</h4>To study the utility of ¹¹C-MET, ¹?F-Fet and ¹?F-FDG for myeloma imaging, time activity curves were compared in various human myeloma cell lines (INA-6, MM1.S, OPM-2) and correlated to cell-biological characteristics, such as marker gene expression and immunoglobulin levels. Likewise, patient-derived CD138? plasma cells were characterized regarding uptake and biomedical features.<h4>Results</h4>Using myeloma cell lines and patient-derived CD138? plasma cells, we found that the relative uptake of ¹¹C-MET exceeds that of ¹?F-FDG 1.5- to 5-fold and that of ¹?F-Fet 7- to 20-fold. Importantly, ¹¹C-MET uptake significantly differed between cell types associated with worse prognosis (e.g. t(4;14) in OPM-2 cells) and indolent ones and correlated with intracellular immunoglobulin light chain and cell surface CD138 and CXCR4 levels. Direct comparison of radiotracer uptake in primary samples further validated the superiority of ¹¹C-MET.<h4>Conclusion</h4>These data suggest that ¹¹C-MET might be a versatile biomarker for myeloma superior to routine functional imaging with ¹?F-FDG regarding diagnosis, risk stratification, prognosis and discrimination of tumor subtypes.