Leukemia-Derived Exosomes Reorganize Bone Marrow Microenvironment In AML

Abstract Increasing evidence suggests that leukemia cells take shelter in the bone marrow (BM) microenvironment (niche), where they hide from chemotherapy and continue to divide. As yet, the identity of niche cells and secreted factors that facilitate leukemia cell growth and assist them in evading chemotherapy is unclear. Further, how leukemia cells alter the bone marrow microenvironment is not known yet. In this study, we provide compelling evidences of a novel role of leukemia-derived exosomes in altering the microenvironment constituents by paracrine mechanisms.As proof-of-concept, we analyzed the cytokines mRNA profiles of primary human and mouse stromal cell co-cultured with primary CD34+CD38- cells from AML patients. Stromal cells co-cultured with leukemia showed increased levels of IL-6, IL-1β, VEGFα, TNF and reduced SDF1 mRNA expression. Similar pattern of gene expression changes were observed from stroma cells co-cultured with leukemia-derived exosomes.By using CFSE labeled exosomes, we observed that leukemia-derived exosomes target marrow stromal and endothelial cells both in-vitro and in-vivo directly. In our in vivo AML model, established using xenografted AML cell lines or primary AML patient samples in Rag2-/- γc-/-mice, we observed expansion of LT-HSC and hematopoietic progenitors compartment. The leukemia animals also showed cellular composition changes in the stromal compartment suggesting osteoblast differentiation was blocked. Interestingly, milder but similar changes were observed in mice treated with leukemia-derived exosomes. Exosomes derived from normal human peripheral blood did not induce significant changes in either hematopoietic or stromal compartments in recipient mice. These data indicate that leukemia cells secrete specialized exosomes to modulate the BM microenvironment. Fluidigm dynamic array analysis of BM stromal cells from leukemic mice revealed that the cell adhesion molecules (NCAM1, VCAM1, CD44, OPN & ICAM1) and factors important for angiogenesis (Angpt1, Angpt 2 &VEGF) were all upregulated in leukemia-modified stromal cells whereas genes important for osteoblast (OCN, OSX), chondrocyte (SOX9) development and HSC maintenance (SDF1 and SCF) were down regulated. These results suggest that leukemia cells can remodel the BM microenvironment by changing the stromal cell composition and influencing expression of important molecular regulators. To evaluate the HSC functions in exosomes-treated mice, we used 5-fluorouracil (5-FU) to suppress hematopoiesis and induce myeloablative stress. Leukemia-derived exosome-pretreated mice succumbed to death earlier compared to the control group (p=0.0001) suggesting that HSCs from leukemia-derived exosome-treated mice may have lower stem cell activity than their counterparts from normal mice. Furthermore, more LT-HSC and hematopoietic progenitors from leukemia-derived exosome-pretreated mice were in active cell cycle (p=0.004 and p=0.01 respectively). These findings support our hypothesis that leukemia cells/exosomes directly or indirectly through leukemia-modified niche, altered the HSCs physiological and quiescence properties. Next we analyzed the ability of leukemia-modified niche to support the normal hematopoiesis. We co-cultured freshly sorted normal CD45.2 LT-HSCs (LSK CD150+CD48-Flk2-) with leukemia cells/exosomes pre-treated stroma cells for 48 hours and transplanted the co-cultured HSC into irradiated CD45.1 mice. 18 weeks after transplantation, we observed a significantly decreased engraftment of the HSCs co-cultured with leukemic cells/exosomes stroma compared with the HSCs co-cultured with normal stroma (p=0.003). Finally, leukemia engrafted better and developed more rapidly (p=0.0026) in mice that received leukemia-derived exosomes pre-treatment. These data suggest that changes induced by leukemia-derived exosomes in the BM niche accelerate leukemia progression and decrease their ability to support HSCs. Collectively, our data demonstrate that the leukemia cells manipulate the bone marrow microenvironment, partly through leukemia-derived exosomes, to suppress the normal hematopoiesis and facilitate growth of the leukemic progeny. Disclosures: No relevant conflicts of interest to declare.

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An Adapting Bone Marrow Niche Creates a Nurturing Environment for Hematopoiesis during Immune Thrombocytopenia Progression

Blood ◽

10.1182/blood-2019-129795 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 222-222

Author(s):

Oliver Herd ◽

Maria Abril Arredondo Garcia ◽

James Hewitson ◽

Karen Hogg ◽

Saleni Pravin Kumar ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Stromal Cell ◽

Cell Subset ◽

Fold Increase ◽

Bone Marrow Microenvironment ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Vessel Area ◽

Chronic Itp

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterised by low platelet counts (<100 x 109/L) and manifests as a bleeding tendency. The demand on hematopoiesis is elevated in chronic ITP, where sustained platelet destruction mediated by an activated immune system is likely to cause considerable stress on progenitor populations. Intriguingly, this increased stress does not appear to result in functional exhaustion, as chronic ITP patients do not present with pancytopenia. By using a novel murine model of chronic ITP, generated by injecting mice with anti-CD41 antibody (ITP group) or IgG (control group) every 48hrs for 4 weeks, we aimed to define the effect of chronic ITP on hematopoietic progenitors and to elucidate the mechanisms behind the preservation of hematopoiesis. The relative numbers of hematopoietic progenitors in mice with chronic ITP vs controls were analysed by flow cytometry and their fitness was assessed by measuring their relative ability to reconstitute the hematopoietic system of lethally irradiated recipients. There was a significant increase in all hematopoietic progenitors analysed in ITP: 2.96-fold increase in multipotent progenitors, 4.66-fold increase in short-term hematopoietic stem cells (ST-HSCs) and 4.93-fold increase in long-term hematopoietic stem cells (LT-HSCs), which led to an increased ability of ITP donor bone marrow to reconstitute irradiated recipients. The results indicate that chronic ITP drives LT-HSCs out of quiescence and causes increased differentiation into committed progenitors in order to meet the increased demand in platelet production. In support of this, increased megakaryopoiesis was observed in chronic ITP, with a 60.5% increase in the number of megakaryocytes observed in bone marrow sections. Interestingly, similar to the clinical manifestation of ITP, we observed no change in levels of circulating TPO in our ITP model. Next, the effect of chronic ITP on the bone marrow microenvironment was determined due to its essential role in the support and maintenance of hematopoiesis. Histological analysis of bone marrow from mice with chronic ITP (Figure 1) revealed a 66.7% increase in the numbers of LepR+/ Cxcl12-DsRed stromal cells. LepR+/ Cxcl12-DsRed stromal cells are a well characterised stromal cell subset, known to be essential for maintenance and retention of HSCs in the bone marrow microenvironment. During chronic ITP, this stromal cell subset maintained their classically defined perivascular location and retained their ability to produce high levels of hematosupportive cytokines (Cxcl12 and Kitl). Chronic ITP was associated with a significant increase in total bone marrow expression (Cxcl12=2.39-fold increase; Kitl=1.71-fold increase), pointing to perivascular stromal cell expansion as being the source of increased local hematopoietic support. Analysis of the bone marrow vascular network revealed that the average vessel area was increased in chronic ITP (54.3% increase), whilst the number of vessels remained unchanged implying that the marrow sinusoids are vasodilated. We hypothesise that an increase in blood vessel area would aid the extravasation of circulating HSCs back into the bone marrow microenvironment where they would contribute to hematopoiesis. By developing an accurate mouse model of chronic ITP, we have identified key alterations in HSCs and the bone marrow microenvironment. Our data clearly demonstrates that in chronic ITP, HSCs are driven out of quiescence and expand in number in order to contribute to the increased demand for hematopoiesis. Furthermore, the bone marrow microenvironment adapts to this increased differentiation pressure on HSCs by creating a hematosupportive, quiescence promoting environment through the expansion of bone marrow stromal cells, and an increase in blood vessel area. Disclosures No relevant conflicts of interest to declare.

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TGF-β Neutralizing Antibody 1D11 Inhibits LIF-JAK-Stat3 Signaling and Enhances Cytarabine Induced Apoptosis in AML Cells in Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v118.21.926.926 ◽

2011 ◽

Vol 118 (21) ◽

pp. 926-926

Author(s):

Yoko Tabe ◽

Yuexi Shi ◽

Zhihong Zeng ◽

Linhua Jin ◽

Yixin Zhou ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Molecular Mechanisms ◽

Leukemia Cell ◽

Neutralizing Antibody ◽

Leukemia Cells ◽

U937 Cells ◽

Induced Apoptosis ◽

Cells Cultured

Abstract Abstract 926 We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al.,Br J Haematol.2008). Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of leukemic BM microenvironment that promote leukemia cell survival and chemoresistance. Our preliminary data indicate that TGF-β neutralizing antibody 1D11 (Genzyme) prevents accumulation of AML cells in a quiescent G0 state under co-culture condition with BM-derived mesenchymal stromal cells (MSC) (Jin et al., ASH abstract 2010). In turn, the chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. In this study, we investigated the anti-leukemic effects and molecular mechanisms of action of TGF-β neutralizing antibody 1D11 under hypoxic conditions. We further investigated the anti-leukemic efficacy of 1D11 combined with CXCR4 antagonist plerixafor in the in vivo leukemia models. AML cells (MV4;11 and U937) were propagated under 1% O2 for at least 14 days to assure their sustained proliferation and survival. Isotype control antibody 13C4 combined with ara-C induced no significant change in apoptosis or cell cycle progression. In MV4;11 cells cultured with 2ng/mL rhTGF-β1, 1D11 (10 μg/mL) induced only minimal apoptosis by itself, yet enhanced low-dose cytarabine (AraC, 0.5 μM) induced apoptosis. This effect was more prominent under hypoxia compared to normoxia (% of subG1 fraction, 21% O2: ara-C, 2.6 ± 0.2%, ara-C + 1D11, 10.8 ± 2.5%, p=0.03; 1% O2: ara-C, 11.3 ± 2.7%, AraC + 1D11, 21.4 ± 0.5%, p=0.001). 1D11 with ara-C abrogated rhTGFβ1-induced accumulation of cells in G0/G1 phase (21% O2; cont, 73.8 ± 4.1, rhTGFβ, 82.2 ± 3.2, rhTGFβ + AraC, 65.4 ± 2.5, rhTGFβ + AraC + 1D11, 50.3 ± 1.9, p=0.001: in 1% O2; cont, 71.8 ± 1.3, rhTGFβ, 85.4 ± 1.4, rhTGFβ + AraC, 79.3 ± 5.1, rhTGFβ + AraC + 1D11, 67.1 ± 4.0, p = 0.03). The anti-leukemic efficacy of 1D11 was next examined in an in vivo leukemia model. 1D11 administered at 5 mg/kg IP every other day in combination with ara-C (50 mg/kg IP weekly) decreased leukemia burden of nude mice injected with Baf3/ITD-luciferase leukemia cells (p=0.002). Administration of small molecule CXCR4 inhibitor plerixafor, which successfully diminished cell migration to CXCL12 in vitro, in combination with 1D11 decreased leukemia burden in vivo (p=0.05), and co-administration of ara-C, plerixafor and 1D11 was most effective (bioluminescence intensity, ×107 photons/sec) control, 1.2 ± 0.2; ara-C, 0.94 ± 0.3; plerixafor + 1D11, 0.56 ± 0.1; plerixafor + 1D11 + ara-C, 0.23 ± 0.09, p=0.003). We next examined the molecular mechanisms responsible for chemosensitization through blockade of TGFβ with 1D11. Treatment with rhTGF-β1 induced upregulation of p21 expression as well as pro-survival phosphorylation of Stat3 in MV4;11 and U937 cells, and these effects were abrogated by 1D11. Knock-down of Stat3 by siRNA increased apoptosis induction in U937 cells cultured in the presence of rhTGFβ1. Notably, 4-fold upregulation of the established TGFβ target, leukemia inhibitory factor (LIF) gene mRNA, was observed after rhTGF-β1 treatment and this was reversed by 1D11. These results indicate that 1D11 inhibits rhTGF-β1-induced autocrine stimulation of pro-survival LIF-JAK-Stat3 signal transduction pathway in AML cells. In summary, blockade of TGF-β by 1D11, and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment. These findings warrant further investigations in human clinical trials. Disclosures: Konopleva: Genzyme: Research Funding.

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Disulfiram, an clinically used anti-alcoholism drug has the potential to target acute myeloid leukemia cells in a copper-dependent manner in vivo

Blood ◽

10.1182/blood.v122.21.5040.5040 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5040-5040

Author(s):

Bing Xu ◽

Rongwei Li ◽

Huijuan Dong ◽

Feili Chen ◽

Yuejian Liu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Specific Antigen ◽

Leukemia Cell ◽

Leukemia Cells ◽

Control Group ◽

Acute Myeloid

Abstract Background Disulfiram(DS), an old drug clinically used for alcoholism, was reported to have antitumor effects, recent studies have found that Copper(Cu) can significantly enhance the DS-induced cell death in vitro in a variety of tumor cells. Our previous studies also demonstrated that disulfiram/copper (DS/Cu) couldtarget human leukemia cell lines(like KG1α,Molt4) through the activation of JNK, in vitro. However, there is few report about the ability of DS/Cu in killing cancer cells in vivo. Aims This study aims to explore the effect of DS/Cu on acute myeloid leukemia cell line KG1αin vivo and clarify the underlining mechanism. Methods 6-8 week old female NOD/SCID mice were sublethally irradiated with 2Gy X-ray the day before transplantation, followed by intravenous injection of KG1α cells (1×107 cells) suspended in 0.2 mL of PBS. 5 weeks after transplantation mice were randomly divided into three treatment groups: vehicle (0.9% saline), a combination of DS and Cu daily for 2 weeks, Ara-C alone twice before killing. Mice were sacrificed after 2 weeks treatment with tissues of spleen, liver, bone marrow being observed using histopathology method to detect the invasion of leukemia. The DS/Cu-induced p-c-jun activation was also examined by western blot using tissues of spleen, liver, bone marrow. Statistical analysis was carried out with one-way ANOVA to assess statistical significance (*p < 0.05). Results 4 weeks after transplantation, mice were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, just like suffered from leukemia. What’s more, immature blasts like morphology similar to KG1α were found in the peripheral blood of the mice(11%±3.41). All the mice were sacrificed after 2 weeks treatment, mice in control group were observed with slightly larger spleen and liver with the morphology of invasion of leukemia such as a granular appearance than the other two groups. Histopathology examination showed that leukemia cells infiltrate liver, spleen and bone marrow, and the immunohistochemistry examination found that the leukemia cells in spleen, liver and bone marrow expressed human specific antigen CD45 with the highest expression level in the control group. Moreover, solid tumor could be observed in the peritoneal cavity of two mice in the control group with expression of human specific antigen CD45detected by immunohistochemistry examination. Western blot in this study showed DS/Cu complex induced phosphorylation of c-Jun expression in the spleen, liver and bone marrow. Conclusion DS/Cu complex could effectively target the acute myeloid leukemia cells in the acute leukemia NOD/SCID mice while inhibiting the invasion of leukemia to some extent, and the activation of JNK might play a functional role in DS/Cu mediated antileukemic effects. Disclosures: No relevant conflicts of interest to declare.

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Impairment of AML Engraftment to the Bone Marrow Niche Following Inhibition of TGF-Beta Signaling

Blood ◽

10.1182/blood.v124.21.4372.4372 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4372-4372 ◽

Cited By ~ 3

Author(s):

Ashley Hamilton ◽

Katie Foster ◽

Dominique Bonnet

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Microenvironment ◽

Laminin Receptor ◽

Cytokine Signaling ◽

Bone Marrow Niche ◽

Tgf Beta ◽

Stromal Compartment ◽

Β Receptor

Abstract The cell fate of the HSC to either self-renew or differentiate is controlled by a complex interplay between cell-intrinsic and -extrinsic regulatory signals generated by the surrounding bone marrow microenvironment called the HSC niche. A balance exists within the “cross talk” between HSCs and the niche, which allows HSC dormancy, activation and differentiation. Any alterations of this balance may lead to uncontrolled cellular proliferation and ultimately the promotion of leukemia. However, it remains to be determined exactly how the hematopoietic microenvironment contributes to the deregulation of normal hematopoiesis and/or promotes the maintenance of leukemia cells as a “leukemic niche”. To investigate this, we have now performed micro-array analysis of MS5 stromal cells that were co-cultured with a panel of leukemic cell lines and acute myeloid leukemia (AML) patient samples. The most significantly up-regulated pathways as compared to MS5 cells cultured alone involved cytoskeleton remodeling, cell cycle, cell adhesion and development through cytokine signaling. Since transcript and protein levels of number of effectors of the TGF-beta (TGF-β) signaling pathway were up-regulated in the stroma co-cultured with leukemic cells, we next investigated inhibition of this pathway using a specific inhibitor of TGF-β receptor kinase, SB-431542 (10µM). Treatment with the inhibitor significantly reduced the cell number and increased the levels of apoptosis in the AML cells co-cultured on stromal cells, whilst having mininal effect on normal cells. Treatment with SB-431542 (10mg/kg), also significantly reduced the level of AML cell engraftment on treatment in vivo (n=3) (untreated- 68.65±6.95; 56.15±22.85; 84.35±5.75 and SB-treated- 45.5±11.6; 30.5±19.6; 54.1±4.9). In order to inhibit TGF-β signaling more specifically within the stromal compartment, we next used shRNA against TGF-β Receptor II (TGFBR2) in MS5 stromal cells and co-cultured them with AML cells within 3D scaffold models (n=4), which were implanted in vivo. A significant reduction in engraftment was observed as compared to controls (shRNA control- 64.65±32.65; 87.7±7.2; 23.55±4.35; 49.65±33.65 and TGFBR2 knockdown- 20.2±3; 62.95±4.05; 15.7±1.5; 20.385±17.415). The co-culturing of normal cord blood CD34+ or mononuclear cells on the TGFBR2 knockdown stroma had no significant effect both in vitro and in vivo (n=3). To investigate whether TGF-β inhibition had an effect on the interaction of AML cells to the niche, we used intravital microscopy to track the cells live in vivo. HL60 (AML cell line) cells were labeled with 2µM CFSE and pretreated ±SB-431542 (10µM) on stroma, before being sorted and transplanted into immunodeficient mice. Distance to the calvaria was measured at 16 hours and we observed that the SB-431542-treated cells were positioned significantly further away from the bone surface as compared to untreated control (p=0.0001). Since the TGF-β inhibited cells appeared to have impaired ability to adhere to the bone marrow, we next investigated the relationship between extracellular matrix molecules and TGF-β signaling. We saw that stromal cells that were co-cultured with AML cells had a significantly increased expression of laminins A1, A5, B1 and G1. This effect could be recapitulated by treatment of naïve stromal cells with TGF-β2 and 3. We also observed a reciprocal decrease in expression of laminins following both treatment of AML-stromal co-cultures with SB-431542 and within TGFBR2 knockdown stroma. Furthermore, we saw an increase in the laminin receptor, integrin alpha-6 (CD49f), in AML cells treated with TGF-β 1, 2 and 3 and a reciprocal decrease following treatment with SB-431542, thereby, indicating that the abrogation of this signaling axis may be, at least, partially responsible for the impaired engraftment of AML cells to their niche following inhibition of the TGF-β pathway. These data thus highlight the potential for the development of therapies directed at modifying the bone marrow microenvironment. Disclosures No relevant conflicts of interest to declare.

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Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist CTCE-9908 Enhances Chemotherapy-Induced Apoptosis in AML

Blood ◽

10.1182/blood.v112.11.2415.2415 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2415-2415

Author(s):

Hongbo Lu ◽

Zhihong Zeng ◽

Yuexi Shi ◽

Sergej Konoplev ◽

Donald Wong ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Leukemia Cell ◽

Bone Marrow Microenvironment ◽

Cell Interactions ◽

Leukemic Cells ◽

Dependent Manner ◽

Induced Apoptosis ◽

Dose Dependent

Abstract The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells towards the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva, Leukemia 2002). Using a peptide analog of SDF-1α designated CTCE-9908, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that CTCE-9908 significantly inhibits SDF-1α-induced migration of U937 (43% inhibition) and OCI-AML3 cells (40% inhibition) in a dose-dependent manner. In three of the four primary AML samples which expressed CXCR4 on cell surface and migrated in response to SDF-1α, 50 μg/ml CTCE-9908 reduced SDF-1α-induced migration of leukemic blasts (60%, 19% and 50% inhibition respectively). In in vitro co-culture systems, stromal cells significantly protected OCI-AML3 cells from chemotherapy induced apoptosis [no MS-5, 75.2±5.2% annexinV(+); with MS-5, 59±1.1% annexinV(+)]. Western blot analysis revealed that CTCE-9908 inhibits Akt and Erk phosphorylation in a dose-dependent manner in the OCI-AML3 cell line stimulated by SDF-1α. Blockade of CXCR4 expression with CTCE-9908 markedly abrogated the protective effects of stromal cells on OCI-AML3 [Ara-C, 59±1.1% annexinV(+); Ara-C + CTCE-9908, 76.9±1.35 annexinV(+)]. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in four out of five primary AML samples with surface expression of functional CXCR4 (mean increase, 25.1±9.3% compared to chemotherapy alone). In vivo, subcutaneous administration of 1.25mg CTCE-9908 induced mobilization of leukemic cells from primary AML patient transplanted into NOD/Scid-IL2Rγ-KO mice (from 15% to 27% circulating leukemic cells 1 hour post CTCE-9908 injection). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis via retention of leukemic cells in the bone marrow microenvironment niches. Disruption of these interactions by the potent CXCR4 inhibitor CTCE-9908 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interaction. Based on these observations, in vivo experiments are ongoing to characterize the efficacy of chemotherapy combined with CTCE-9908.

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Acute myeloid leukemia induces protumoral p16INK4a-driven senescence in the bone marrow microenvironment

Blood ◽

10.1182/blood-2018-04-845420 ◽

2019 ◽

Vol 133 (5) ◽

pp. 446-456 ◽

Cited By ~ 17

Author(s):

Amina M. Abdul-Aziz ◽

Yu Sun ◽

Charlotte Hellmich ◽

Christopher R. Marlein ◽

Jayna Mistry ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Stromal Cell ◽

Cell Senescence ◽

Senescent Phenotype ◽

Age Related ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. With increasing age, tissues accumulate senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of a set of proinflammatory cytokines, chemokines, and growth factors, collectively known as the senescence-associated secretory phenotype (SASP). Here, we report that AML blasts induce a senescent phenotype in the stromal cells within the BM microenvironment and that the BM stromal cell senescence is driven by p16INK4a expression. The p16INK4a-expressing senescent stromal cells then feed back to promote AML blast survival and proliferation via the SASP. Importantly, selective elimination of p16INK4a+ senescent BM stromal cells in vivo improved the survival of mice with leukemia. Next, we find that the leukemia-driven senescent tumor microenvironment is caused by AML-induced NOX2-derived superoxide. Finally, using the p16-3MR mouse model, we show that by targeting NOX2 we reduced BM stromal cell senescence and consequently reduced AML proliferation. Together, these data identify leukemia-generated NOX2-derived superoxide as a driver of protumoral p16INK4a-dependent senescence in BM stromal cells. Our findings reveal the importance of a senescent microenvironment for the pathophysiology of leukemia. These data now open the door to investigate drugs that specifically target the “benign” senescent cells that surround and support AML.

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Erythroid Differentiation Regulator 1 (ERDR1) From Nurse-Like Cells Promotes the Survival of Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v116.21.1372.1372 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1372-1372

Author(s):

Hendrik W. Van Deventer ◽

Robert Mango ◽

Jonathan Serody

Keyword(s):

Bone Marrow ◽

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Mesenchymal Cells ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Wild Type

Abstract Abstract 1372 Background: Chemotherapy resistance in chronic lymphocytic leukemia (CLL) can be mediated by anti-apoptotic signals produced by stromal or nurse-like cells. Developing strategies to overcome this resistance is hindered by the lack of suitable “stromal” targets responsible for these signals. We have discovered that erythroid differentiation regulator 1 (ERDR1) may be a candidate target for such a strategy. In this study, we show Erdr1 is generated by several stromal cell types including bone marrow stromal cells, fibrocytes, and nurse-like cells. Furthermore, inhibition of stroma-generated Erdr1 results in increased apoptosis of co-cultured CLL cells. Methods/Results: We initially identified Erdr1 on an Affymetrix array that compared the gene expression of wild type and CCR5-/- mice with pulmonary metastasis. The increased expression of Erdr1 in the wild type mice was particularly pronounced in the pulmonary mesenchymal cells. Therefore, these cells were transfected with one of two shRNAs (shRNA #9 or shRNA#11) and the survival of these cells was compared with mesenchymal cells transfected with a non-targeted control vector. After 15 days in culture, the control cells expanded normally; however, no significant expansion was seen in either the shRNA#9 or shRNA#11 transfected cells. These differences in cellular expansion were associated with differences in apoptosis. 21.4+1.6% of the Erdr1 knockdown cells were annexin V+ compared to 11.2+1.9% of the non-targeted control (p<0.03). Using GFP as a marker for transfection, we were also able to show that knockdown of Erdr1 increased the apoptosis of surrounding non-transfected mesenchymal cells. Thus, Erdr1 is a critical protein for the survival of stromal cells. Further analysis of the mesenchymal cell subpopulations revealed the greatest expression of Erdr1 in the CD45+, thy1.1+/− fibrocytes. When compared to CD45- fibroblasts, the fibrocytes expressed CCR5 and increased Erdr1 expression by 14.2+/−2.9 fold when treated with the CCR5 ligand CCL4. Given the similarities between fibrocytes and nurse-like cells, we went on to measure the effect of Erdr1 inhibition on CLL cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. These clones were then co-cultured with primary CLL cells. At 96 hours, leukemia cells co-cultured with the control lines had expanded by 1.33 + 0.9 compared to 0.74 + 0.22 fold in the knock-down lines (p<0.03). As before, the lack of cellular expansion was associated with an increase in apoptosis. To further show the relevance of these findings to CLL, we demonstrated that human fibrocytes and nurse-like cells expressed mRNA and protein for ERDR1 in all patient samples tested. Implications for the treatment of human disease: Our data demonstrate that ERDR1 is a critically important protein for the survival of nurse-like cells. These data suggest that targeting ERDR1 or the upstream pathway through CCR5 might be a novel approach for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

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Stroma-Mediated Chemotherapy Resistance in Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v118.21.242.242 ◽

2011 ◽

Vol 118 (21) ◽

pp. 242-242

Author(s):

Xin Long ◽

Tsz-Kwong Man ◽

Michele S. Redell

Keyword(s):

Bone Marrow ◽

Conditioned Medium ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Chemotherapy Resistance ◽

Physical Contact ◽

Leukemia Cells ◽

Soluble Factors

Abstract Abstract 242 Overall objective: AML is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Once a child relapses, the chance of survival is very low. Therefore new, rational therapies for AML are desperately needed. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in AML cells, which could potentially lead to new therapies for AML. Methods, Results & Conclusions: We used two human bone marrow stromal cell lines, HS-5 and HS-27 for our studies. Both provide physical contact with AML cells, while HS-5 cells secrete many more cytokines and growth factors than HS-27 stromal cells. To verify the difference between HS-5 and HS-27 in their secreted soluble factors, both stroma-conditioned media were harvested and soluble factors were quantified by multiplex cytokine assay for 42 individual soluble factors. We detected 23 factors in HS-5 conditioned medium, including G-CSF, IL-6, and MCP-3 at very high levels. HS-27-conditioned medium contained only a few cytokines at similar levels as HS-5, e.g., VEGF and Fractalkine. Next, we performed co-culture experiments to determine the ability of each stromal cell line to confer resistance to chemotherapy. Human AML cell lines (NB-4, THP-1 and Kasumi-1) were cultured alone or co-cultured with HS-5 or HS-27 cells, and treated with etoposide, mitoxantrone or cytarabine for 48 hours. Cells were then harvested and labeled with annexin V-FITC. Stromal cells were identifiable by stable mOrange expression, and the percentage of apoptotic AML cells (FITC positive and mOrange negative) was determined by FACS. Both HS-5 (p<0.001) and HS-27 (p<0.05) cells protected NB-4 and THP-1 cells from etoposide-induced apoptosis (apoptosis rate at the 3 uM dose: 86.6±1.4% NB-4 alone vs. 33.9±2.9% with HS-5 vs 60.7±2.5% with HS-27). The results with THP-1 were similar to NB-4. Using the same method, we demonstrated that both stromal cells protected NB-4 and THP-1 from the toxic effects of all three chemotherapy agents; Kasumi-1 were resistant to all three agents, even when cultured alone. To delineate if the protection induced by stromal cells against chemotherapy was dependent on adhesion pathways and/or soluble factors, we performed Transwell co-culture assays. Different from regular co-culture, there is no physical contact between AML and stromal cells, while soluble factors secreted by stromal cells can reach AML cells. In the absence of physical contact, both stromal cells provided little protection for NB-4 and THP-1 against etoposide and cytarabine; while both NB-4 and THP-1 were still protected against mitoxantrone. Those results suggest that the protection provided by both stromal cells against etoposide and cytarabine mostly relies on cell-cell contact; as for mitoxantrone, soluble factors secreted by both stromal cells seem more important. Surprisingly, HS-5 and HS-27 provided similar degrees of protection against all three chemotherapies. To discover genes in AML cells that are induced by interaction with stromal cells and may contribute to chemotherapy resistance, oligonucleotide microarray analysis was done using total RNA extracted from NB-4 and THP-1 cells cultured alone or co-cultured with stromal cells. We found that 43 genes were upregulated by HS-5, and over 1000 genes were either up- or down-regulated by HS-27. Among them, eighteen genes were upregulated by both stromal cell lines. Since HS-5 and HS-27 provided similar degrees of protection against chemotherapy, those eighteen commonly upregulated genes are likely to be important for stroma-induced chemotherapy resistance. Excitingly, seven out of those eighteen genes, e.g., including CYR61, CAV1, TM4SF1, have been reported to contribute to chemotherapy resistance in various cancer types. Further studies are underway to determine if those genes are responsible for stroma-induced chemotherapy resistance. This study suggests that distinct pathways in the microenvironment mediate resistance to different chemotherapy drugs. Elucidating the precise drug-specific mechanisms involved is likely to result in promising combination therapies to reduce chemotherapy resistance and relapse, and thereby improve survival for children with AML. Disclosures: No relevant conflicts of interest to declare.

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ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Blood ◽

10.1182/blood.v120.21.893.893 ◽

2012 ◽

Vol 120 (21) ◽

pp. 893-893

Author(s):

Po Yee Mak ◽

Duncan H Mak ◽

Yuexi Shi ◽

Vivian Ruvolo ◽

Rodrigo Jacamo ◽

...

Keyword(s):

Bone Marrow ◽

Leukemia Cell ◽

Leukemia Cells ◽

Apoptosis Resistance ◽

Control Mscs ◽

Extrinsic Apoptosis ◽

Induced Apoptosis ◽

And Control

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

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Bone Marrow Stroma Protects Myeloma Cells from Cytotoxic Damage Via Induction of the Oncoprotein MUC1

Blood ◽

10.1182/blood.v124.21.3378.3378 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3378-3378

Author(s):

Michal Bar-Natan ◽

Katarina Luptakova ◽

Maxwell Douglas Coll ◽

Dina Stroopinsky ◽

Hasan Rajabi ◽

...

Keyword(s):

Bone Marrow ◽

Drug Resistance ◽

Cell Viability ◽

Cell Line ◽

Stromal Cells ◽

Stromal Cell ◽

Fold Increase ◽

Bone Marrow Microenvironment ◽

Muc1 Expression ◽

Myeloma Cells

Abstract Introduction : Stromal cells in the bone marrow microenvironment of patients with multiple myeloma (MM) are thought to play a vital role in promoting cell growth and protection from cytotoxic injury. Targeting of stromal-myeloma cell interactions to enhance anti-myeloma treatment represents a promising therapeutic strategy. The MUC1 oncoprotein is a critical oncoprotein that is expressed in the majority of primary myeloma cells and regulates downstream pathways such as NFkB and β-catenin/wnt that modulate myeloma growth and survival. Inhibition of MUC1 via a cell penetrating peptide (GO-203) that blocks down stream signaling reverses resistance to bortezomib (BZT). Herein we studied the influence of bone marrow stromal cells (BMSC) on MUC1 expression on MM cells, and its link to drug resistance. Methods and Results : Coculture of MM human cell lines (RPMI and U266) with a stromal cell line (HS-5), resulted in an upregulation of MUC1 expression as determined by an approximately 2 fold increase in the mean fluorescent intensity (MFI) of MUC1 as measured by flow cytometry. Similar findings were observed following coculture of MM cells with stromal cells isolated from primary bone marrow mononuclear cells (BMSC) of MM patients. Stromal cell mediated upregulation of MUC1 expression was subsequently confirmed by Western blot analysis. Patient derived MM cells were also noted to increase their MUC1 expression 2.9 fold when co-cultured with stroma (HS-5 cell line). MUC1 expression was also increased following coculture of MM cells with stromal cells in transwell plates, suggesting the effect was mediated by soluble factors not requiring cell-cell contact. Consistent with these findings, we demonstrated that addition of recombinant IL-6, a stromal cell derived cytokine, to MM cells resulted in a 2 fold increase in MFI of MUC1 expression. Moreover, coculture of MM cells with IL-6 neutralizing antibodies abrogated the effect of BMSC on MUC1 expression. These results suggest that stromal cell secretion of IL-6 plays a role in upregulation of the oncoprotein MUC1 on MM cells. We subsequently evaluated the effect of stromal cell induction of MUC1 expression on resistance to anti-myeloma agents. Increased MUC1 expression following coculture of MM cells with BMSC was associated with a higher level of resistance to BTZ (20nM), resulting in 48% less cell death by CellTiter-Glo and annexin/propidium iodide (PI) staining. Conversely, we demonstrated that silencing of MUC1 expression using a lentiviral siRNA resulted in enhanced sensitivity to anti-myeloma agents. Cell viability in MUC1 silenced as compared to wild type RPMI cells decreased by 18%, 43%, and 50% when treated with 10mg/ml cyclophosphamide (Cy), 5nM BZT, and 0.1mM melphalan, respectively. MUC1 silenced U266 cells demonstrated a decrease in cell viability by 24%, 34%, and 45% when treated with 10mg/ml Cy, 5nM BZT, and 1mM lenalidomide respectively. Similarly, exposure of primary MM cells to the MUC1 inhibitor GO-203 resulted in enhanced MM cell sensitivity to bortezomib and cyclophosphamide evidenced by a 60% and 39% decrease in cell viability respectively, compared to each drug alone. Conclusions : Our results delineate one of the mechanisms by which the bone marrow microenvironment confers drug resistance in MM. MM cells co-cultured with BMSC have enhanced expression of MUC1, mediated by IL-6 secretion. Overexpression in turn confers MM cell resistance to standard anti-myeloma agents. Importantly inhibition of MUC1 via silencing of expression or exposure to a small molecule inhibitor can overcome drug resistance to known anti-myeloma drugs, providing the rationale for clinical evaluation of combination therapy. Disclosures Kufe: Genus Oncology: Consultancy, Equity Ownership.

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