Pyrvinium Pamoate Use in a B cell Acute Lymphoblastic Leukemia Model of the Bone Tumor Microenvironment.

PURPOSE: Pyrvinium pamoate (PP) is an anthelmintic drug that has been found to have anti-cancer activity in several cancer types. In the present study, we evaluated PP for potential anti-leukemic activity in B cell acute lymphoblastic leukemia (ALL) cell lines, in an effort to evaluate the repurposing potential of this drug in leukemia. METHODS: ALL cells were treated with PP at various concentrations to determine its effect on cell proliferation. Metabolic function was tested by evaluating Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). Lastly, 3D spheroids were grown, and PP was reformulated into nanoparticles to evaluate distribution effectiveness. RESULTS: PP was found to inhibit ALL proliferation, with varied selectivity to different ALL cell subtypes. We also found that PP's cell death activity was specific for leukemic cells, as primary normal immune cells were resistant to PP-mediated cell death. Metabolic studies indicated that PP, in part, inhibits mitochondrial oxidative phosphorylation. To increase the targeting of PP to a hypoxic bone tumor microenvironment (BTME) niche, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. CONCLUSION: We have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells and can be effectively formulated into a nanoparticle delivery system to target the BTME.

The MitoNEET Ligand NL-1 Mediates Antileukemic Activity in Drug-Resistant B-Cell Acute Lymphoblastic Leukemia.

Disease relapse in B-cell acute lymphoblastic leukemia (ALL), either due to development of acquired resistance after therapy or because of de novo resistance, remains a therapeutic challenge. In the present study, we have developed a cytarabine (Ara-C)-resistant REH cell line (REH/Ara-C) as a chemoresistance model. REH/Ara-C 1) was not crossresistant to vincristine or methotrexate; 2) showed a similar proliferation rate and cell surface marker expression as parental REH; 3) demonstrated decreased chemotaxis toward bone marrow stromal cells; and 4) expressed higher transcript levels of cytidine deaminase (CDA) and mitoNEET (CISD1) than the parental REH cell line. Based on these findings, we tested NL-1, a mitoNEET inhibitor, which induced a concentration-dependent decrease in cell viability with a comparable IC50 value in REH and REH/Ara-C. Furthermore, NL-1 decreased cell viability in six different ALL cell lines and showed inhibitory activity in a hemosphere assay. NL-1 also impaired the migratory ability of leukemic cells, irrespective of the chemoattractant used, in a chemotaxis assay. More importantly, NL-1 showed specific activity in inducing death in a drug-resistant population of leukemic cells within a coculture model that mimicked the acquired resistance and de novo resistance observed in the bone marrow of relapsed patients. Subsequent studies indicated that NL-1 mediates autophagy, and inhibition of autophagy partially decreased NL-1-induced tumor cell death. Finally, NL-1 showed antileukemic activity in an in vivo mouse ALL model. Taken together, our study demonstrates that mitoNEET has potential as a novel antileukemic drug target in treatment refractory or relapsed ALL.

Novel compounds that target lipoprotein lipase and mediate growth arrest in acute lymphoblastic leukemia.

Over the past decade, the therapeutic strategies employed to treat B-precursor acute lymphoblastic leukemia (ALL) have been progressively successful in treating the disease. Unfortunately, the treatment associated dyslipidemia, either acute or chronic, is very prevalent and a cause for decreased quality of life in the surviving patients. To overcome this hurdle, we tested a series of cylopropanecarboxamides, a family demonstrated to target lipid metabolism, for their anti-leukemic activity in ALL. Several of the compounds tested showed anti-proliferative activity, with one, compound 22, inhibiting both Philadelphia chromosome negative REH and Philadelphia chromosome positive SupB15 ALL cell division. The novel advantage of these compounds is the potential synergy with standard chemotherapeutic agents, while concomitantly blunting the emergence of dyslipidemia. Thus, the cylopropanecarboxamides represent a novel class of compounds that can be potentially used in combination with the present standard-of-care to limit treatment associated dyslipidemia in ALL patients.

Bone marrow osteoblast vulnerability to chemotherapy.

Osteoblasts are a major component of the bone marrow microenvironment, which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN), and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. As one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy-induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post-transplantation.

Chemotherapeutic Activity of Pitavastatin in Vincristine Resistant B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (ALL) is derived from an accumulation of malignant, immature B cells in the bone marrow and blood. Relapse due, in part, to the emergence of tumor cells that are resistant to front line standard chemotherapy is associated with poor patient outcomes. This challenge highlights the need for new treatment strategies to eliminate residual chemoresistant tumor cells. Based on the use of pitavastatin in acute myeloid leukemia (AML), we evaluated its efficacy in an REH ALL cell line derived to be resistant to vincristine. We found that pitavastatin inhibited the proliferation of both parental and vincristine-resistant REH tumor cells at an IC50 of 449 nM and 217 nM, respectively. Mitochondrial bioenergetic assays demonstrated that neither vincristine resistance nor pitavastatin treatment affected cellular oxidative phosphorylation, beta-oxidation, or glycolytic metabolism in ALL cells. In a co-culture model of ALL cells with bone marrow stromal cells, pitavastatin significantly decreased cell viability more robustly in the vincristine-resistant ALL cells compared with their parental controls. Subsequently, NSG mice were used to develop an in vivo model of B-cell ALL using both parental and vincristine-resistant ALL cells. Pitavastatin (10 mg/kg i.p.) significantly reduced the number of human CD45+ REH ALL cells in the bone marrow of mice after 4 weeks of treatment. Mechanistic studies showed that pitavastatin treatment in the vincristine-resistant cells led to apoptosis, with increased levels of cleaved PARP and protein-signaling changes for AMP-activated protein kinase/FoxO3a/Puma. Our data suggest the possible repurposing of pitavastatin as a chemotherapeutic agent in a model of vincristine-resistant B-cell ALL.

Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts.

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.

Pitavastatin Is Anti-Leukemic in a Bone Marrow Microenvironment Model of B-Lineage Acute Lymphoblastic Leukemia.

The lack of complete therapeutic success in the treatment of B-cell acute lymphoblastic leukemia (ALL) has been attributed, in part, to a subset of cells within the bone marrow microenvironment that are drug resistant. Recently, the cholesterol synthesis inhibitor, pitavastatin (PIT), was shown to be active in acute myeloid leukemia, prompting us to evaluate it in our in vitro co-culture model, which supports a chemo-resistant ALL population. We used phospho-protein profiling to evaluate the use of lipid metabolic active compounds in these chemo-resistant cells, due to the up-regulation of multiple active survival signals. In a co-culture with stromal cells, a shift towards anabolic processes occurred, which was further confirmed by assays showing increased lipid content. The treatment of REH leukemia cells with pitavastatin in the co-culture model resulted in significantly higher leukemic cell death than exposure to the standard-of-care chemotherapeutic agent, cytarabine (Ara-C). Our data demonstrates the use of pitavastatin as a possible alternative treatment strategy to improve patient outcomes in chemo-resistant, relapsed ALL.

A prospective study of filgrastim pharmacokinetics in morbidly obese patients compared with non-obese controls.

INTRODUCTION: Filgrastim is a human granulocyte colony-stimulating factor (G-CSF). There are limited data on dosing filgrastim in obesity. The objective of this study was to compare filgrastim pharmacokinetic parameters for morbidly obese and non-obese patients after a single subcutaneous dose of filgrastim dosed per actual body weight. METHODS: This prospective, matched-pair study (NCT01719432) included patients ≥18 years of age, receiving filgrastim at 5 µg/kg with a weight >190% of their ideal body weight (IBW) for "morbidly obese" patients or within 80%-124% of IBW for matched-control patients. The control group was prospectively matched for age (within 10 years), degree of neutropenia, and gender. Filgrastim doses were not rounded to vial size, to allow more accurate assessment of exposure. Blood samples were collected at 0 (prior to dose), 2, 4, 6, 8, 12, and 24 h after the first subcutaneous administration of filgrastim. RESULTS: A total of 30 patients were enrolled in this prospective pharmacokinetic study, with 15 patients assigned to each arm. Non-compartmental analysis showed that the systemic clearance (Cl) was 0.111 ± 0.041 ml/min in the morbidly obese group versus 0.124 ± 0.045 ml/min in the non-obese group (p = 0.44). Additionally, the mean area under the curve (AUC0-24h ) was 49.3 ± 13.9 ng/ml × min in the morbidly obese group versus 46.3 ± 16.8 ng/mL x min in the non-obese group (p = 0.6). No differences were seen in maximum concentrations (Cmax ) between the two groups (morbidly obese: 48.1 ± 14.7 ng/ml vs. non-obese: 49.2 ± 20.7 ng/ml (p = 0.87)). The morbidly obese group had a numerically higher, but not statistically significant, increase in time to maximum concentration (Tmax ) compared to the non-obese group (544 ± 145 min vs 436 ± 156 min (p = 0.06), respectively). CONCLUSION: Calculating subcutaneous filgrastim doses using actual body weight appears to produce similar systemic exposure in morbidly obese and non-obese patients with severe neutropenia.

Respiratory syncytial virus infection in human bone marrow stromal cells.

Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children. The pathophysiology of this infection in the respiratory system has been studied extensively, but little is known about its consequences in other systems. We studied whether RSV infects human bone marrow stromal cells (BMSCs) in vitro and in vivo, and investigated whether and how this infection affects BMSC structure and hematopoietic support function. Primary human BMSCs were infected in vitro with recombinant RSV expressing green fluorescent protein. In addition, RNA from naive BMSCs was amplified by PCR, and the products were sequenced to confirm homology with the RSV genome. The BMSC cytoskeleton was visualized by immunostaining for actin. Finally, we analyzed infected BMSCs for the expression of multiple cytokines and chemokines, evaluated their hematopoietic support capacity, and measured their chemotactic activity for both lymphoid and myeloid cells. We found that BMSCs support RSV replication in vitro with efficiency that varies among cell lines derived from different donors; furthermore, RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. RSV infection disrupted cytoskeletal actin microfilaments, altered cytokine/chemokine expression patterns, decreased the ability of BMSCs to support B cell maturation, and modulated local chemotaxis. Our data indicate that RSV infects human BMSCs in vitro, and this infection has important structural and functional consequences that might affect hematopoietic and immune functions. Furthermore, we have amplified viral RNA from naive primary BMSCs, suggesting that in vivo these cells provide RSV with an extrapulmonary target.

Co-culture model of B-cell acute lymphoblastic leukemia recapitulates a transcription signature of chemotherapy-refractory minimal residual disease.

B-cell acute lymphoblastic leukemia (ALL) is characterized by accumulation of immature hematopoietic cells in the bone marrow, a well-established sanctuary site for leukemic cell survival during treatment. While standard of care treatment results in remission in most patients, a small population of patients will relapse, due to the presence of minimal residual disease (MRD) consisting of dormant, chemotherapy-resistant tumor cells. To interrogate this clinically relevant population of treatment refractory cells, we developed an in vitro cell model in which human ALL cells are grown in co-culture with human derived bone marrow stromal cells or osteoblasts. Within this co-culture, tumor cells are found in suspension, lightly attached to the top of the adherent cells, or buried under the adherent cells in a population that is phase dim (PD) by light microscopy. PD cells are dormant and chemotherapy-resistant, consistent with the population of cells that underlies MRD. In the current study, we characterized the transcriptional signature of PD cells by RNA-Seq, and these data were compared to a published expression data set derived from human MRD B-cell ALL patients. Our comparative analyses revealed that the PD cell population is markedly similar to the MRD expression patterns from the primary cells isolated from patients. We further identified genes and key signaling pathways that are common between the PD tumor cells from co-culture and patient derived MRD cells as potential therapeutic targets for future studies.

Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (ALL) affects both pediatric and adult patients. Chemotherapy resistant tumor cells that contribute to minimal residual disease (MRD) underlie relapse and poor clinical outcomes in a sub-set of patients. Targeting mitochondrial oxidative phosphorylation (OXPHOS) in the treatment of refractory leukemic cells is a potential novel approach to sensitizing tumor cells to existing standard of care therapeutic agents. In the current study, we have expanded our previous investigation of the mitoNEET ligand NL-1 in the treatment of ALL to interrogate the functional role of the mitochondrial outer membrane protein mitoNEET in B-cell ALL. Knockout (KO) of mitoNEET (gene: CISD1) in REH leukemic cells led to changes in mitochondrial ultra-structure and function. REH cells have significantly reduced OXPHOS capacity in the KO cells coincident with reduction in electron flow and increased reactive oxygen species. In addition, we found a decrease in lipid content in KO cells, as compared to the vector control cells was observed. Lastly, the KO of mitoNEET was associated with decreased proliferation as compared to control cells when exposed to the standard of care agent cytarabine (Ara-C). Taken together, these observations suggest that mitoNEET is essential for optimal function of mitochondria in B-cell ALL and may represent a novel anti-leukemic drug target for treatment of minimal residual disease.

Effect of morphine sulfate on neonatal neutrophil chemotaxis.

BACKGROUND: Opioids have been increasingly used for pain control in the neonatal intensive care unit. Data from adult human studies have demonstrated suppressive effects of morphine sulfate on the immune system, owing in part to its inhibition of chemotaxis. OBJECTIVE: To study the effect of morphine exposure on chemotaxis of newborn neutrophils compared with adult neutrophils. METHODS: Blood samples were collected from adult controls and from the umbilical cord of healthy full-term newborns. Neutrophils were isolated and then exposed to morphine sulfate. Chemotaxis assays were performed using interleukin (IL)-8 as the chemoattractant. The migrated neutrophils were quantitated by flow cytometry. IL-8 receptor expression was evaluated by staining with an anti-IL-8 receptor-specific antibody. Chemotaxis and IL-8 receptor expression were compared between newborn and adult neutrophils. RESULTS: There was no difference in random migration between adult (n=10) and newborn neutrophils (n=14). IL-8 efficiently induced chemotaxis of both adult and newborn neutrophils, although newborn neutrophils exhibited significantly decreased chemotaxis compared with adult neutrophils: 389+/-197 newborn cells versus 731+/-190 adult cells (P=0.025). Exposure to morphine sulfate did not decrease chemotaxis of adult neutrophils but did modestly impair chemotaxis of newborn neutrophils. After exposure to morphine sulfate, adult neutrophils showed no difference in IL-8 receptor expression, whereas newborn neutrophils expressed fewer IL-8 receptors. CONCLUSIONS: Newborn neutrophils had reduced chemotaxis toward IL-8. Exposure to morphine sulfate further decreased their chemotactic function. The differential effect may be explained in part by the reduction of IL-8 receptors of newborn neutrophils after morphine exposure.

Combination of cabazitaxel and plicamycin induces cell death in drug resistant B-cell acute lymphoblastic leukemia.

Bone marrow microenvironment mediated downregulation of BCL6 is critical for maintaining cell quiescence and modulating therapeutic response in B-cell acute lymphoblastic leukemia (ALL). In the present study, we have performed a high throughput cell death assay using BCL6 knockdown REH ALL cell line to screen a library of FDA-approved oncology drugs. In the process, we have identified a microtubule inhibitor, cabazitaxel (CAB), and a RNA synthesis inhibitor, plicamycin (PLI) as potential anti-leukemic agents. CAB and PLI inhibited cell proliferation in not only the BCL6 knockdown REH cell line, but also six other ALL cell lines. Furthermore, combination of CAB and PLI had a synergistic effect in inhibiting proliferation in a cytarabine-resistant (REH/Ara-C) ALL cell line. Use of nanoparticles for delivery of CAB and PLI demonstrated that the combination was very effective when tested in a co-culture model that mimics the in vivo bone marrow microenvironment that typically supports ALL cell survival and migration into protective niches. Furthermore, exposure to PLI inhibited SOX2 transcription and exposure to CAB inhibited not only Mcl-1 expression but also chemotaxis in ALL cells. Taken together, our study demonstrates the utility of concomitantly targeting different critical regulatory pathways to induce cell death in drug resistant ALL cells.

Chemotherapy disrupts activity of translational regulatory proteins in bone marrow stromal cells.

OBJECTIVE: Bone marrow stromal cell function is a critical influence on hematopoietic reconstitution following progenitor or stem cell transplantation. Stromal cells support hematopoietic cell migration, survival, and proliferation. We have previously reported that stromal cell matrix metalloproteinase-2 (MMP-2) is necessary for optimal support of pro-B-cell chemotaxis through its regulation of stromal cell-derived factor-1 (CXCL12) release. Following exposure to the topoisomerase II inhibitor, etoposide (VP-16), stromal cell MMP-2 protein expression is reduced. The current study investigated the mechanism by which VP-16 may alter translation of MMP-2 in bone marrow stromal cells. MATERIALS AND METHODS: Bone marrow stromal cells were exposed to chemotherapeutic agents etoposide, melphalan, and 4-hydroperoxycyclophosphamide (4HC) and evaluated for MMP-2 expression by enzyme-linked immunosorbent assay and support of pro-B-cell chemotaxis by chemotaxis assay. Western blot analyses were completed to evaluate phosphorylation of stromal cell translational regulatory proteins 4E binding protein-1 (4EBP-1), P70(S6K), and S6 or MMP-2 in the presence of chemotherapy, or the chemical inhibitors rapamycin or LY294002. RESULTS: Rapid dephosphorylation of 4EBP-1, P70(S6K), and S6 following VP-16 exposure was observed, consistent with blunted translational efficiency. We also observed that inhibition of stromal cell mammalian target of rapamycin with rapamycin, or phosphatidylinositol 3 kinase with LY294002, resulted in inhibition of stromal cell MMP-2 protein. In addition we found that the chemotherapeutic agents melphalan and 4HC disrupt bone marrow stromal cell MMP-2 protein expression and support of chemotaxis. CONCLUSIONS: These data suggest that one mechanism by which chemotherapy may alter stromal cells of the bone marrow microenvironment is through disrupted translation of proteins.

MMP-2 is required for bone marrow stromal cell support of pro-B-cell chemotaxis.

OBJECTIVE: We have previously demonstrated that bone marrow stromal cells (BMSCs) exposed to etoposide (VP-16) have reduced support of CXCR4(+) cell chemotaxis and diminished stromal cell derived factor-1 (CXCL12) in the supernatants. Based on the identification of CXCL12 as a matrix metalloproteinase-2 (MMP-2) substrate, we investigated potential dysregulation of MMP-2 expression or activity in chemotherapy-treated BMSCs. METHODS: BMSCs exposed to VP-16 were evaluated for MMP-2 expression by gelatin zymography, ELISA, and western blot. Chemotaxis assays were completed to evaluate pro-B cell chemotaxis toward either MMP-2(-/-) BMSCs or BMSCs exposed to MMP-2 inhibitors. RESULTS: BMSC exposure to VP-16 resulted in an immediate, transient, increase in MMP-2, followed by reduced MMP-2 protein expression. MMP-2 reduction correlated with diminished CXCL12 protein and reduced support of pro-B cell chemotaxis. BMSCs derived from MMP-2 knockout mice had less chemotactic support of CXCR4(+) cells than wild-type controls. Inhibition of BMSC MMP-2 activity by OA-Hy also reduced chemotactic support and CXCL12 protein detected in BMSC supernatants. VP-16-induced reduction of BMSC support of hematopoietic cell migration was restored by supplementing cultures with recombinant MMP-2 protein. CONCLUSIONS: These data suggest that MMP-2 is sensitive to chemotherapy-induced stress, and may regulate BMSC support of pro-B cell chemotaxis. Increased MMP-2 expression during the acute phase of chemotherapy exposure potentially inactivates CXCL12. Subsequently, chronic exposure to chemotherapy, with the associated downregulation of MMP-2, interrupts CXCL12 release from the extracellular matrix, also blunting BMSC support of pro-B cell migration.

BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy.

The bone marrow niche has a significant impact on acute lymphoblastic leukemia (ALL) cell phenotype. Of clinical relevance is the frequency with which quiescent leukemic cells, in this niche, survive treatment and contribute to relapse. This study suggests that marrow microenvironment regulation of BCL6 in ALL is one factor that may be involved in the transition between proliferative and quiescent states of ALL cells. Utilizing ALL cell lines, and primary patient tumor cells we observed that tumor cell BCL6 protein abundance is decreased in the presence of primary human bone marrow stromal cells (BMSC) and osteoblasts (HOB). Chemical inhibition, or shRNA knockdown, of BCL6 in ALL cells resulted in diminished ALL proliferation. As many chemotherapy regimens require tumor cell proliferation for optimal efficacy, we investigated the consequences of constitutive BCL6 expression in leukemic cells during co-culture with BMSC or HOB. Forced chronic expression of BCL6 during co-culture with BMSC or HOB sensitized the tumor to chemotherapy induced cell death. Combination treatment of caffeine, which increases BCL6 expression in ALL cells, with chemotherapy extended the event free survival of mice. These data suggest that BCL6 is one factor, modulated by microenvironment derived cues that may contribute to regulation of ALL therapeutic response.

Chemotherapy-induced Dkk-1 expression by primary human mesenchymal stem cells is p53 dependent.

Mesenchymal stem cells (MSCs) are abundant throughout the body and regulate signaling within tumor microenvironments. Wnt signaling is an extrinsically regulated pathway that has been shown to regulate tumorigenesis in many types of cancer. After evaluating a panel of Wnt activating and inhibiting molecules, we show that primary human MSCs increase the expression of Dkk-1, an inhibitor of Wnt signaling, into the extracellular environment following chemotherapy exposure in a p53-dependent manner. Dkk-1 has been shown to promote tumor growth in several models of malignancy, suggesting that MSC-derived Dkk-1 could counteract the intent of cytotoxic chemotherapy, and that pharmacologic inhibition of Dkk-1 in patients receiving chemotherapy treatment for certain malignancies may be warranted.

Bone Marrow Microenvironment Niche Regulates miR-221/222 in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) has many features in common with normal B-cell progenitors, including their ability to respond to diverse signals from the bone marrow microenvironment (BMM) resulting in regulation of cell-cycle progression and survival. Bone marrow-derived cues influence many elements of both steady state hematopoiesis and hematopoietic tumor cell phenotypes through modulation of gene expression. miRNAs are one regulatory class of small noncoding RNAs that have been shown to be increasingly important in diverse settings of malignancy. In the current study, miRNA profiles were globally altered in ALL cells following exposure to primary human bone marrow niche cells, including bone marrow stromal cells (BMSC) and primary human osteoblasts (HOB). Specifically, mature miR-221 and miR-222 transcripts were decreased in ALL cells cocultured with BMSC or HOB, coincident with increased p27 (CDKN1B), a previously validated target. Increased p27 protein in ALL cells exposed to BMSC or HOB is consistent with accumulation of tumor cells in the G0 phase of the cell cycle and resistance to chemotherapy-induced death. Overexpression of miR-221 in ALL cells during BMSC or HOB coculture prompted cell-cycle progression and sensitization of ALL cells to cytotoxic agents, blunting the protective influence of the BMM. These novel observations indicate that BMM regulation of miR-221/222 contributes to marrow niche-supported tumor cell quiescence and survival of residual cells. IMPLICATIONS: Niche-influenced miR-221/222 may define a novel therapeutic target in ALL to be combined with existing cytotoxic agents to more effectively eradicate refractory disease that contributes to relapse. Mol Cancer Res; 14(10); 909-19. ©2016 AACR.

In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage.

Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

Modeling Chemotherapy Resistant Leukemia In Vitro.

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.