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.

Tumor microenvironment-targeted nanoparticles loaded with bortezomib and ROCK inhibitor improve efficacy in multiple myeloma.

Drug resistance and dose-limiting toxicities are significant barriers for treatment of multiple myeloma (MM). Bone marrow microenvironment (BMME) plays a major role in drug resistance in MM. Drug delivery with targeted nanoparticles have been shown to improve specificity and efficacy and reduce toxicity. We aim to improve treatments for MM by (1) using nanoparticle delivery to enhance efficacy and reduce toxicity; (2) targeting the tumor-associated endothelium for specific delivery of the cargo to the tumor area, and (3) synchronizing the delivery of chemotherapy (bortezomib; BTZ) and BMME-disrupting agents (ROCK inhibitor) to overcome BMME-induced drug resistance. We find that targeting the BMME with P-selectin glycoprotein ligand-1 (PSGL-1)-targeted BTZ and ROCK inhibitor-loaded liposomes is more effective than free drugs, non-targeted liposomes, and single-agent controls and reduces severe BTZ-associated side effects. These results support the use of PSGL-1-targeted multi-drug and even non-targeted liposomal BTZ formulations for the enhancement of patient outcome in MM.

CXCR4-targeted PET imaging using 64Cu-AMD3100 for detection of Waldenström Macroglobulinemia.

Objective:  Waldenström Macroglobulinemia (WM) is a rare B-cell malignancy characterized by secretion of immunoglobulin M and cancer infiltration in the bone marrow. Chemokine receptor such as CXCR4 and hypoxic condition in the bone marrow play crucial roles in cancer cell trafficking, homing, adhesion, proliferation, survival, and drug resistance. Herein, we aimed to use CXCR4 as a potential biomarker to detect hypoxic-metastatic WM cells in the bone marrow and in the circulation by using CXCR4-detecting radiopharmaceutical.Methods: We radiolabeled a CXCR4-inhibitor (AMD3100) with 64Cu and tested its binding to WM cells with different levels of CXCR4 expression using gamma counter in vitro. The accumulation of this radiopharmaceutical tracer was tested in vivo in subcutaneous and intratibial models using PET/CT scan. In addition, PBMCs spiked with different amounts of WM cells ex vivo were detected using gamma counting.Results: In vitro, 64Cu-AMD3100 binding to WM cell lines demonstrated a direct correlation with the level of CXCR4 expression, which was increased in cells cultured in hypoxia with elevated levels of CXCR4, and decreased in cells with CXCR4 and HIF-1α knockout. Moreover, 64Cu-AMD3100 detected localized and circulating CXCR4high WM cells with high metastatic potential.Conclusions: In conclusion, we developed a molecularly targeted system, 64Cu-AMD3100, which binds to CXCR4 and specifically detects WM cells with hypoxic phenotype and metastatic potential in the subcutaneous and intratibial models. These preliminary findings using CXCR4-detecting PET radiopharmaceutical tracer indicate a potential technology to predict high-risk patients for the progression to WM due to metastatic potential.

Selinexor Overcomes Hypoxia-Induced Drug Resistance in Multiple Myeloma.

Increased levels of the nuclear export protein, exportin 1 (XPO1), were demonstrated in multiple myeloma (MM) patients. Targeting XPO1 with selinexor (the selective inhibitor of nuclear export; SINE compound KPT-330) demonstrates broad antitumor activity also in patient cells resistant to bortezomib; hence, it is a promising target in MM patients. Hypoxia is known to mediate tumor progression and drug resistance (including bortezomib resistance) in MM cells. In this study, we tested the effects of selinexor alone or in combination with bortezomib in normoxia and hypoxia on MM cell survival and apoptosis in vitro and in vivo. In vitro, selinexor alone decreased survival and increased apoptosis, resensitizing MM cells to bortezomib. In vivo, we examined the effects of selinexor alone on tumor initiation and tumor progression, as well as selinexor in combination with bortezomib, on tumor growth in a bortezomib-resistant MM xenograft mouse model. Selinexor, used as a single agent, delayed tumor initiation and tumor progression, prolonging mice survival. In bortezomib-resistant xenografts, selinexor overcame drug resistance, significantly decreasing tumor burden and extending mice survival when combined with bortezomib.

Tariquidar sensitizes multiple myeloma cells to proteasome inhibitors via reduction of hypoxia-induced P-gp-mediated drug resistance.

Multiple myeloma (MM) presents a poor prognosis and high lethality of patients due to development of drug resistance. P-glycoprotein (P-gp), a drug-efflux transporter, is upregulated in MM patients post-chemotherapy and is involved in the development of drug resistance since many anti-myeloma drugs (including proteasome inhibitors) are P-gp substrates. Hypoxia develops in the bone marrow niche during MM progression and has long been linked to chemoresistance. Additionally, hypoxia-inducible transcription factor (HIF-1α) was demonstrated to directly regulate P-gp expression. We found that in MM patients P-gp expression positively correlated with the hypoxic marker, HIF-1α. Hypoxia increased P-gp protein expression and its efflux capabilities in MM cells in vitro using flow cytometry. We reported herein that hypoxia-mediated resistance to carfilzomib and bortezomib in MM cells is due to P-gp activity and was reversed by tariquidar, a P-gp inhibitor. These results suggest combining proteasome inhibitors with P-gp inhibition for future clinical studies.

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.

Tris DBA palladium overcomes hypoxia-mediated drug resistance in multiple myeloma.

Despite recent progress in novel and targeted therapies, multiple myeloma (MM) remains a therapeutically challenging incurable disease. The regulation of important cellular processes and its link to cancer presented Src as an attractive target for MM. We suggest a novel strategy to improve the treatment of MM and overcome the drug resistance for the current therapeutic agents by specific inhibition of Src in MM cells by Tris (Dibenzylideneacetone) dipalladium (Tris DBA). Tris DBA reduces proliferation, induces G1 arrest and apoptosis in MM cells. Tris DBA showed additive effect with proteasome inhibitors reducing proliferation, cell cycle signaling, and increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to proteasome inhibitors by inhibition of HIF1α expression. Moreover, we found that Tris DBA is an effective anti-myeloma agent alone or in combination with other targeted drugs and that it reverses hypoxia-induced drug resistance in myeloma.

Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib.

Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

3D tissue-engineered bone marrow as a novel model to study pathophysiology and drug resistance in multiple myeloma.

PURPOSE: Multiple myeloma (MM) is the second most prevalent hematological malignancy and it remains incurable despite the introduction of several novel drugs. The discrepancy between preclinical and clinical outcomes can be attributed to the failure of classic two-dimensional (2D) culture models to accurately recapitulate the complex biology of MM and drug responses observed in patients. EXPERIMENTAL DESIGN: We developed 3D tissue engineered bone marrow (3DTEBM) cultures derived from the BM supernatant of MM patients to incorporate different BM components including MM cells, stromal cells, and endothelial cells. Distribution and growth were analyzed by confocal imaging, and cell proliferation of cell lines and primary MM cells was tested by flow cytometry. Oxygen and drug gradients were evaluated by immunohistochemistry and flow cytometry, and drug resistance was studied by flow cytometry. RESULTS: 3DTEBM cultures allowed proliferation of MM cells, recapitulated their interaction with the microenvironment, recreated 3D aspects observed in the bone marrow niche (such as oxygen and drug gradients), and induced drug resistance in MM cells more than 2D or commercial 3D tissue culture systems. CONCLUSIONS: 3DTEBM cultures not only provide a better model for investigating the pathophysiology of MM, but also serve as a tool for drug development and screening in MM. In the future, we will use the 3DTEBM cultures for developing personalized therapeutic strategies for individual MM patients.

The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy.

Hypoxia is a non-physiological level of oxygen tension, a phenomenon common in a majority of malignant tumors. Tumor-hypoxia leads to advanced but dysfunctional vascularization and acquisition of epithelial-to-mesenchymal transition phenotype resulting in cell mobility and metastasis. Hypoxia alters cancer cell metabolism and contributes to therapy resistance by inducing cell quiescence. Hypoxia stimulates a complex cell signaling network in cancer cells, including the HIF, PI3K, MAPK, and NFĸB pathways, which interact with each other causing positive and negative feedback loops and enhancing or diminishing hypoxic effects. This review provides background knowledge on the role of tumor hypoxia and the role of the HIF cell signaling involved in tumor blood vessel formation, metastasis, and development of the resistance to therapy. Better understanding of the role of hypoxia in cancer progression will open new windows for the discovery of new therapeutics targeting hypoxic tumor cells and hypoxic microenvironment.

Hypoxia promotes dissemination and colonization in new bone marrow niches in Waldenström macroglobulinemia.

UNLABELLED: Waldenström macroglobulinemia, a rare and indolent type of non-Hodgkin lymphoma, is characterized by widespread lymphoplasmacytic B cells in the bone marrow. Previous studies have shown that hypoxic conditions play a key role in the dissemination of other hematologic malignancies. In this study, the effect of hypoxia was tested on the progression and spread of Waldenström macroglobulinemia. Interestingly, tumor progression correlated with hypoxia levels in Waldenström macroglobulinemia cells and other cells in the bone marrow and correlated with the number of circulating tumor cells in vivo. Mechanistic studies demonstrated that hypoxia decreased cell progression and cell cycle, did not induce apoptosis, and reduced the adhesion between Waldenström macroglobulinemia cells and bone marrow stroma, through downregulation of E-cadherin expression, thus explaining increased egress of Waldenström macroglobulinemia cells to the circulation. Moreover, hypoxia increased the extravasation and homing of Waldenström macroglobulinemia cells to new bone marrow niches in vivo, by increased CXCR4/SDF-1-mediated chemotaxis and maintaining the VLA4-mediated adhesion. Re-oxygenation of hypoxic Waldenström macroglobulinemia cells enhanced the rate of proliferation and cell cycle progression and restored intercellular adhesion between Waldenström macroglobulinemia cells and bone marrow stroma. This study suggests that targeting hypoxic response is a novel strategy to prevent dissemination of Waldenström macroglobulinemia. IMPLICATIONS: This study provides a better understanding of the biology of dissemination of Waldenström macroglobulinemia and opens new windows for investigation of new therapeutic targets in Waldenström macroglobulinemia based on tumor hypoxia mechanisms.

The role of hypoxia and exploitation of the hypoxic environment in hematologic malignancies.

Tumor hypoxia is a well-described phenomenon during the progression of solid tumors affecting cell signaling pathways and cell metabolism; however, its role in hematologic malignancies has not been given the same attention in the literature. Therefore, this review focuses on the comparative differences between solid and hematologic malignancies with emphasis on the role of hypoxia during tumorigenesis and progression. In addition, contribution of the bone marrow and angiogenic environment are also discussed. Insight is provided into the role of hypoxia in metastatic spread, stemness, and drug resistance in hematologic conditions. Finally, emerging therapeutic strategies such as small-molecule prodrugs and hypoxia-inducible factor (HIF) targeting approaches are outlined to combat hypoxic cells and/or adaptive mechanisms in the treatment of hematologic malignancies.

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.

Molecularly targeted therapies in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that remains incurable because most patients will eventually relapse or become refractory to the treatments. Although the treatments have improved, the major problem in MM is the resistance to therapy. Novel agents are currently in development for the treatment of relapsed/refractory MM, including immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, cell signaling targeted therapies, and strategies targeting the tumor microenvironment. We have previously reviewed in detail the contemporary immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies therapies for MM. Therefore, in this review, we focused on the role of molecular targeted therapies in the treatment of relapsed/refractory multiple myeloma, including cell signaling targeted therapies (HDAC, PI3K/AKT/mTOR, p38 MAPK, Hsp90, Wnt, Notch, Hedgehog, and cell cycle) and strategies targeting the tumor microenvironment (hypoxia, angiogenesis, integrins, CD44, CXCR4, and selectins). Although these novel agents have improved the therapeutic outcomes for MM patients, further development of new therapeutic agents is warranted.

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.

Novel tumor suppressor function of glucocorticoid-induced TNF receptor GITR in multiple myeloma.

Glucocorticoid-induced TNF receptor (GITR) plays a crucial role in modulating immune response and inflammation, however the role of GITR in human cancers is poorly understood. In this study, we demonstrated that GITR is inactivated during tumor progression in Multiple Myeloma (MM) through promoter CpG island methylation, mediating gene silencing in primary MM plasma cells and MM cell lines. Restoration of GITR expression in GITR deficient MM cells led to inhibition of MM proliferation in vitro and in vivo and induction of apoptosis. These findings were supported by the presence of induction of p21 and PUMA, two direct downstream targets of p53, together with modulation of NF-κB in GITR-overexpressing MM cells. Moreover, the unbalanced expression of GITR in clonal plasma cells correlated with MM disease progression, poor prognosis and survival. These findings provide novel insights into the pivotal role of GITR in MM pathogenesis and disease progression.