Potentiation of the Effects of Nilotinib by Combination with Plerixafor in a Mouse Model of BCR-ABL-Positive Residual Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2737-2737
Author(s):  
Ellen L Weisberg ◽  
Paul W. Manley ◽  
Andrew L. Kung ◽  
James D. Griffin
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Hematopoietic Cells ◽  
Tumor Burden ◽  
Leukemic Cells ◽  
High Dose

Abstract Abstract 2737 Background: It has been shown that a small, residual pool of leukemic CD34+ progenitor cells can survive in the marrow microenvironment of chronic myeloid leukemia (CML) patients after years of kinase inhibitor treatment. Bone marrow stroma contributes to the expansion and proliferation of both transformed as well as normal hematopoietic cells, and has been implicated in the long-term survival of leukemic cells. We previously demonstrated the stromal protection of leukemic cells from the anti-proliferative effects of nilotinib, and identified stromal-derived viability factors, including IL-6 and GM-CSF, as possibly mediating stromal protection of tyrosine kinase inhibitor-treated leukemic cells (Weisberg et al. Mol Cancer Ther 2008;7:1121). Additionally, we have found high leukemia burden in the tissues of nilotinib-treated mice that have significant sources of hematopoiesis-promoting stroma, suggesting that significant reservoirs for tumor growth may be tissues that are able to support normal and malignant hematopoietic stem cell development. These studies revealed a leukemia distribution pattern consistent with that observed in imatinib or nilotinib-treated patients. One strategy for overriding stromal-mediated chemoresistance is the use of inhibitors of the stroma-derived factor (SDF-1a) receptor, CXCR4, which mediates the migration of hematopoietic cells to the bone marrow and plays a key role in leukemic cell-stromal cell interactions. Studies have shown small-molecule CXCR4 inhibitors, such as plerixafor, to be effective in mobilizing hematopoietic cells from bone marrow and enhancing chemotherapy- and tyrosine kinase inhibitor-induced apoptosis of bone marrow stroma-protected leukemic cells in vitro and in vivo. Thus, imatinib has been shown to up-regulate CXCR4, which induces CML migration to the bone marrow microenvironment and leads to stroma-mediated chemoresistance of quiescent CML progenitor cells. Methodology: Utilizing a functional in vivo assay system that allows monitoring of the growth of progressive disease, as well as baseline level (“residual” disease) resulting from treatment with a moderate-to-high dose of nilotinib, we investigated the ability of stem cell mobilization to enhance the efficacy of nilotinib by suppressing leukemia recurrence following nilotinib treatment. Specifically, 32D.p210-luc+ cells were injected into the tail-vein of mice, which were then imaged 10 days later to determine baseline bioluminescence and quantify tumor-burden. Mice were subsequently treated by oral gavage for 10 days with nilotinib (75 mg/kg qd) and reimaged (“Induction Phase” of treatment); mice were at this stage considered to have minimal residual disease, with reduced tumor burden > 2 logs. Mice were then divided into four treatment groups (“Consolidation Phase” of treatment addressing minimum residual disease) with similar mean bioluminescence (n=8 or 9 per group): Group 1: Vehicle (PEG300 po), Group 2: plerixafor (5 mg/kg sq qd), Group 3: nilotinib (75mg/kg po qd), and Group 4: Combination (plerixafor+nilotinib). Results: Nilotinib was highly efficacious in reducing disease burden in leukemia-engrafted mice. However, with continued treatment, animals developed resistance to nilotinib with increasing disease burden despite continued therapy. Although plerixafor had no single-agent activity, combination with nilotinib significantly delayed time to relapse, and significantly prolonged survival when compared to nilotinib monotherapy (p<0.0001). Since plerixafor had no monotherapeutic efficacy, there results demonstrate that plerixafor, at a well-tolerated dose, acts synergistically with nilotinib to suppress the growth of 32D.p210 leukemia. Conclusion: We demonstrated the ability of plerixafor to delay the onset of recurring BCR-ABL-positive disease in mice carrying an extremely low tumor burden following treatment with a moderate-to-high dose of nilotinib. These results support the idea of using stem cell mobilization in conjunction with targeted tyrosine kinase inhibition to override drug resistance and suppress or eradicate residual disease. Disclosures: Manley: Novartis Pharma AG: Employment. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Griffin:Novartis Pharmaceuticals: Consultancy, Research Funding.

Inhibiting the IGF-1 Receptor Tyrosine Kinase with Picropodophillin: An In Vitro and In Vivo Study in the 5T33MM Mouse Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 640-640
Author(s):  
Karin Vanderkerken ◽  
Eline Menu ◽  
Thomas Stromberg ◽  
Hendrik De Raeve ◽  
Kewal Asosingh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Microvessel Density ◽  
Kinase Inhibitor ◽  
High Dose

Abstract Multiple myeloma (MM) represents a B-cell malignancy, characterized by monoclonal proliferation of plasma cells in the bone marrow (BM) and is associated with osteolysis and angiogenesis. Insulin-like growth factor-1 (IGF-1), produced by the BM stromal cells, has been described as an important factor in the survival, proliferation and migration of MM cells. The latter process is involved in the homing of the MM cells to the BM. IGF-1 also induces VEGF secretion by the MM cells, thus stimulating angiogenesis in the BM. As IGF-1 is a pleiotropic factor in MM, therapeutic strategies targeting the IGF-1R may be effective as anti-tumor treatments. In this work we investigated the effect of an IGF-1 receptor tyrosine kinase inhibitor (picropodophyllin or PPP1) in the murine, syngeneic 5T33MM model of multiple myeloma. This mouse model is representative for the human disease and can combine in vitro and in vivo studies. We first investigated the effects of PPP on the MM cells in vitro. We and others have previously demonstrated that IGF-1 induced ERK activation, involved in VEGF secretion and proliferation. When the 5T33MM cells were preincubated with 1microM PPP, Western blot analysis demonstrated the blocking of this activation. Furthermore, when the 5T33MM cells were preincubated with PPP for 30 min, IGF-1 induced VEGF secretion and proliferation of the 5T33MM cells were completely blocked. Next, we used the tyrosine kinase inhibitor PPP in vivo. 5T33MM cells were injected intravenously in C57BLKaLwRij mice and the development of the disease was monitored by measuring the serum paraprotein concentration. Mice were either treated with a low (17mM, IP, twice a day) or a high dose of PPP (50mM, IP, twice a day) or with the vehicle (DMSO/oil 9/1) from the day of injection with 5T33MM onward. At week 3, vehicle controls showed signs of morbidity and were sacrificed. The presence of tumor was measured by assessing serum paraprotein concentrations and determining the proportion of idiotype positive cells in the BM by flow cytometry. Angiogenesis was assessed by measuring the microvessel density on CD31 stained paraffin sections. The tumor burden in the bone marrow in the PPP treated mice was 77% lower than in vehicle treated animals (p< 0,0001) and the serum paraprotein concentration was 90% lower (p< 0,0001). The microvessel density in the BM of the PPP treated group was reduced by 60% (p< 0,02). In a separate survival experiment the mice were either treated with the vehicle or with the high dose (50mM) of PPP, from the time of tumor injection. Kaplan-Meier analysis demonstrated a significant increase in survival after treatment with PPP when compared with vehicle (28 vs. 18 days, p<0,001). These data demonstrate that the IGF-1RTK inhibitor PPP possesses strong anti-tumor activity, as demonstrated both in vitro and in vivo in a syngeneic model of multiple myeloma, and may therefore be an effective therapeutic candidate for MM treatment.

scholarly journals Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia

Blood ◽  
2020 ◽  
Vol 136 (2) ◽  
pp. 210-223 ◽  
Author(s):  
Eun Ji Gang ◽  
Hye Na Kim ◽  
Yao-Te Hsieh ◽  
Yongsheng Ruan ◽  
Heather A. Ogana ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Underlying Mechanism ◽  
Conditional Knockout

Abstract Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Preclinical Evaluation of a Bone-Marrow Autograft Culture Procedure for Generating Lymphokine-Activated Killer Cells in Vitro

1992 ◽  
Vol 3 (suppl b) ◽  
pp. 123-127 ◽  
Author(s):  
Hans-Georg Klingemann ◽  
Heather Deal ◽  
Dianne Reid ◽  
Connie J Eaves
Keyword(s):  
Bone Marrow ◽  
Calf Serum ◽  
Cell Activity ◽  
Hematopoietic Cells ◽  
Lak Cells ◽  
High Dose ◽  
Lymphokine Activated Killer Cells ◽  
Lak Cell

Despite the use of high dose chemoradiotherapy for the treatment of acute leukemia. relapse continues to be a major cause of death in patients given an autologous bone marrow transplant. Further augmentation of pretransplant chemotherapy causes life threatening toxicity to nonhematopoietic tissues and the effectiveness of currently available ex vivo purging methods in reducing the relapse rate is unclear. Recently, data from experimental models have suggested that bone marrow-derived lymphokine (IL-2)-activated killer (BM-LAK) cells might be used to eliminate residual leukemic cells both in vivo and in vitro. To evaluate this possibility clinically, a procedure was developed for culturing whole marrow harvests with IL-2 prior to use as autografts, and a number of variables examined that might affect either the generation of BM-LAK cells or the recovery of the primitive hematopoietic cells. The use of Dexter long term culture (LTC) conditions, which expose the cells to horse serum and hydrocortisone. supported LAK cell generation as effectively as fetal calf serum (FCS) -containing medium in seven-day cultures. Maintenance of BM-LAK cell activity after a further seven days of culture in the presence of IL-2 was also tested. As in the clinical setting. patients would receive IL-2 in vivo for an additional week immediately following infusion of the cultured marrow autograft. Generation ofBM-LAK activity was dependent on the presence of IL-2 and could be sustained by further incubation in medium containing IL-2. Primitive hematopoietic cells were quantitated by measuring the number of in vitro colony-forming progenitors produced after five weeks in secondary Dexter-type LTC. Maintenance of these 'LTC-initiating cells' was unaffected by lL-2 in the culture medium. These results suggest that LAK cells can be generated efficien tly in seven-day marrow autograft cultures containing IL-2 under conditions that allow the most primitive human hematopoietic cells currently detectable to be maintained.

scholarly journals The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission

Haematologica ◽  
2020 ◽  
Vol 106 (1) ◽  
pp. 111-122 ◽  
Author(s):  
Sandrine Jeanpierre ◽  
Kawtar Arizkane ◽  
Supat Thongjuea ◽  
Elodie Grockowiak ◽  
Kevin Geistlich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Stromal Cells ◽  
Kinase Inhibitor ◽  
Bmp Signaling ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Exhaustion of Donor Hematopoietic Stem Cells in Lethally-Irradiated Hosts Can Be Sbstantially Mitigated by Targeting p18INK4C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1200-1200
Author(s):  
Hui Yu ◽  
Youzhong Yuan ◽  
Xianmin Song ◽  
Feng Xu ◽  
Hongmei Shen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Total Period ◽  
Over Expression

Abstract Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

High Efficacy and Good Tolerability with Rituximab In Vivo Purging Followed by High Dose Chemotheraphy and Autologous Peripheral Blood Stem Cell Transplantation (APBSCT) in Non-Hodgkin’s Lymphoma (NHL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4626-4626
Author(s):  
Yuankai Shi ◽  
Sheng Yang ◽  
Xiaohong Han ◽  
Peng Liu ◽  
Xiaohui He ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Complete Remission ◽  
Minimal Residual Disease ◽  
Median Time ◽  
Residual Disease ◽  
High Dose ◽  
Platelet Recovery ◽  
Minimal Residual

Abstract Purpose: High-dose chemotherapy (HDC) supported by APBSCT has been shown to be superior to standard therapy in NHL. However, many patients relapse due to minimal residual disease (MRD) in vivo or in the graft. Rituximab has the potential to clear both blood and bone marrow of malignant CD20+ cells, prompting this multicenter trial of in vivo purging with rituximab and HDC with APBSCT in China. Methods: Cyclophosphamide 4g/m2 was used as the mobilization regimen, CY/TBI, BEAM or CBV could be used as HDC at the discretion of the institution. Four infusions of rituximab (375 mg/m2) were given: one day before mobilization, one day before harvesting, one day before transplantation and on day 8 after transplantation. BCL-2/Ig-H translocation was measured as a marker of minimal residual disease in blood or bone marrow before mobilization and during transplantation using real-time quantitative PCR. Results: Thirty-one patients from 12 centers with histologically proven CD20+ NHL (28 aggressive, 3 indolent NHL) were enrolled. Twenty-four patients were previously untreated, and 7 patients had relapsed disease. Median yields of CD34+ cells and mononuclear cells were 5.9×106/kg and 4.4×108 /kg respectively. Median time to recovery of WBC >1.5×109/L, ANC >0.5×109/L and platelets >20×109/L after APBSCT was 10 days in each case. Median time to platelet recovery >50×109/L was 13 days. Generally, this therapeutic strategy was well tolerated with few side effects attribute to rituximab. All patients achieved a complete remission after APBSCT. At a median-follow-up of 12 months, overall survival and progression-free survival (PFS) are 87% and 73% respectively for all patients. In patients with aggressive NHL, overall survival and PFS are 85% and 73% respectively and in indolent NHL are 100% and 67% respectively. PFS and overall survival were slightly higher in previously untreated compared with relapsed patients (88% vs. 83% for PFS, 73% vs. 69% for overall survival). One of five 5 patients who were initially found to be PCR-positive and achieved PCR-negative status subsequently experienced progression accompanied by a return to PCR positivity. The remaining four patients are still in complete remission and are PCR negative. Conclusion: These results suggest that the regimen of rituximab combined with HDCT and APBSCT is effective and well tolerated for the treatment of patients with NHL.

Specialized Bone Marrow Endothelium Defines Microdomains for Tumor and Stem Cell Engraftment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 663-663
Author(s):  
Dorothy A. Sipkins ◽  
Xunbin Wei ◽  
Juwell W. Wu ◽  
Terry K. Means ◽  
Andrew D. Luster ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Cells ◽  
Stem Cell Differentiation ◽  
Multiple Time ◽  
Normal Stem Cell ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Vascular Beds

Abstract The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation and regeneration, yet relatively little is known about the architecture of microenvironments that support malignant proliferation. Using dynamic in vivo confocal and multi-photon imaging, we show that the bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant SDF-1 in discrete, discontinuous areas that localize the homing and early engraftment of both leukemic and normal primitive hematopoietic cells. Real-time imaging of cell-cell interactions in SCID mice bone marrow was performed after injection of fluorescently-labeled leukemic and other malignant cell lines. Progressive scanning and optical sectioning through the marrow revealed the existence of unique, spatially-restricted vascular domains to which the majority of marrow-homing tumor cells rolled and arrested. Serial imaging of mice on days 3 – 14 demonstrated that leukemic (Nalm-6 pre-B ALL) extravasation and early proliferation were restricted to these vascular beds. To define the molecular basis of these homing interactions, in vivo labeling of key vascular cell adhesion molecules and chemokines using fluorescent antibodies was performed. We observed that while ICAM-1, VCAM-1, PECAM-1 and P-selectin were expressed diffusely throughout the marrow vasculature, the expression of E-selectin and the chemokine receptor CXCR4 ligand SDF-1 was distinctly limited to vessels that supported leukemic cell engraftment. In vivo co-localization experiments confirmed Nalm-6 binding was restricted to vascular beds expressing both E-selectin and SDF-1. In functional studies, disruption of E-selection had a modest effect on leukemic homing (<20% diminution), while pharmacologic blockade of CXCR4 decreased Nalm-6 binding to vessels by approximately 80%. To explore the normal function of this vascular niche, we next examined whether benign primitive hematopoietic cells might preferentially home to these same vascular microdomains. Fluorescently-labeled stem and progenitor cells (HSPC) isolated from donor balb/c mice were injected into recipient mice and imaging was performed at multiple time points. HSPC were found to adhere to the BM microvasculature in the same restricted domains. At 70 days post-injection, HSPC had extravasated, were persistent in these perivascular areas and had undergone cell division as assessed by dye dilution. Our findings show that these microdomains serve as vascular portals around which leukemic and hematopoietic stem cells engraft, suggesting that this molecularly distinct vasculature provides both a cancer and normal stem cell niche. Specialized vascular structures therefore appear to delineate a stem cell microenvironment that is exploited by malignancy.

scholarly journals Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment

Blood ◽  
2012 ◽  
Vol 119 (21) ◽  
pp. 4971-4980 ◽  
Author(s):  
Ye Chen ◽  
Rodrigo Jacamo ◽  
Yue-xi Shi ◽  
Rui-yu Wang ◽  
Venkata Lokesh Battula ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
In Vivo Model ◽  
Hematopoietic Microenvironment ◽  
Cell Engraftment

Abstract The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.

scholarly journals Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 778-778
Author(s):  
Zhihong Zeng ◽  
Yuexi Shi ◽  
Twee Tsao ◽  
Yihua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Signaling Pathways ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Mtor Signaling ◽  
Leukemic Cells ◽  
Survival Signaling

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

scholarly journals Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 361-369 ◽  
Author(s):  
PE Funk ◽  
PW Kincade ◽  
PL Witte
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Factor C

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

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