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More than half of T-ALL patients harbor gain-of-function mutations in the intracellular domain of Notch1. Diffuse infiltration of the bone marrow commonly occurs in T-ALL and relapsed B-ALL patients, and is associated with worse prognosis. However, the mechanism of leukemia outgrowth in the marrow and the resulting biological impact on hematopoiesis are poorly understood. Here, we investigated targetable cellular and molecular abnormalities in leukemia marrow stroma responsible for the suppression of normal hematopoiesis using a T-ALL mouse model and human T-ALL xenografts. We found that actively proliferating leukemia cells inhibited normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the peri-vascular region. In addition, leukemia development was accompanied by the suppression of the endosteum-lining osteoblast population. We further demonstrate that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. Notch blockade reversed attenuated HSPC cycling, leukemia-associated abnormal blood lineage distribution and thrombocytopenia as well as recovered osteoblast and HSPC abundance and improved the hematopoietic-supportive functions of osteoblasts. Finally, we confirmed that reduced osteoblast frequency and enhanced Notch signaling were also features of the marrow stroma of human ALL tissues. Collectively, our findings suggest that therapeutically targeting the leukemia-infiltrated hematopoietic niche may restore HSPC homeostasis and improve the outcome of ALL patients.
Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children and young adolescence, with up to 20–25% of patients experience relapse with bone marrow being the most common site of all relapses (1). Patients experience bone marrow relapse often have worse outcomes (2, 3). Compared to B-ALL, T-ALL patients have a higher relative risk of death, often showing diffuse infiltration of the bone marrow by immature T cell lymphoblasts prior to T-ALL propagation, and a strong propensity to infiltrate the CNS (4). A better understanding of interplay between ALL and the marrow microenvironment during disease progression would help identify new therapy targets and improve patient outcome.
Recent studies have shown progress in our understanding of leukemia microenvironment. These have revealed contributions of both osteoblasts in B-ALL xenografts and the peri-vascular niche to the progression of AML development in NOD-SCID/IL2Rγnull mice (5, 6). Osteoblasts lining the endosteum play a critical role in hematopoietic stem cell (HSC) homing and maintenance (7–9). A direct interaction between HSCs and the sinusoidal endothelial cells in the peri-vascular niche also supports HSC proliferation (10–13), while CXCL12-abundant reticular (CAR) cells promote HSC retention and proliferation (12). However, little is known about alterations and regulations of T-ALL microenvironment.
Approximately 50–70% of T-ALL harbor “gain-of-function” mutations in the intracellular domain of Notch1 (ICN1) (14). Notch1 is a member of a family of heterodimeric receptors. Ligand binding initiates successive proteolytic cleavage of Notch, which culminate in the release of the ICN1 and the formation of a transcriptional activation complex leading to the activation of downstream targets (15, 16). Notch1 mutations in T-ALL drive leukemia transformation characterized by the expansion of an immature population of T progenitors. A mouse model of T-ALL is achieved in marrow progenitors via retroviral transduction and expression of ICN1 that drives thymus-independent induction of T cell leukemia (17–19). Using this model of T-ALL, we report here that when ICN1-expressing marrow progenitors and wild type (WT) marrow cells were co-transplanted into WT mice, WT marrow cells displayed a disease stage-dependent reduction of the hematopoietic stem and progenitor cells (HSPCs), a prominent suppression of B lymphopoiesi, and thrombocytopenia. Using this mouse model of T-ALL and studies of human T-ALL xenografts, we show that leukemia-induced suppression of hematopoiesis involves reconstruction of the bone marrow microenvironment by hijacking the proliferative vascular space and by repressing the endosteal/osteoblastic niche.
The animal research described in this manuscript was approved by the Institutional Animal Care and Use Committee. Animals used in this study are 8–16 weeks old C57BL/6 (Ly5.2) and B6.SJL-Ptprca Pep3b/BoyJ (Ly5.1) and were maintained as described (20, 21). NSG mice of 8–12 weeks old were from Jackson Laboratory.
Retroviral (pMIG-eGFP-ICN1 provided by Drs John Lowe, Jon Aster and Andrew Weng) transfection of progenitors-enriched bone marrow cells after 5-FU treatment was performed as described (21). Forty-eight hours after infection, 2–5×105 cells along with 2×105 WT cells (Ly5.1) were transferred into lethally irradiated (10.5 Gy) WT recipients (Ly5.1). Recipients were monitored for leukemia development weekly. Secondary leukemia was generated in non-irradiated mice by i.v. injecting primary leukemia splenocytes (2–5×105).
Isolation of HSPCs, analysis of megakaryocyte/erythroid progenitors and Ki67 staining were performed as described (22). Osteoblast sorting and cell surface CXCL12 staining were performed using a modified procedure (22, 23). Megakaryocyte progenitors and erythroid progenitor markers were analyzed by PE-anti-CD41, PE-anti-TER119, and APC-anti-CD71 of LSK cells.
Lineage-depleted progenitors or LSKs were cultured with OP9 for 8 or 4 days in the presence of SCF (25 ng/ml), IL-7 (5 ng/mL) and Flt3 ligand (5 ng/mL), and immunophenotyped as described previously (24). Confluent MC3T3 (provided by Dr. Yibin Kang; re-tested for osteoblast differentiation) cells were co-cultured with 0.5–1×106 total marrow cells from ICN1-mice or pMIG-mice in the presence or absence of 10µM dibenzazepine (DBZ, Selleckchem, Houston, TX) for 72 hours followed by qRT-PCR, or 48 hours followed by co-culture with LSK cells (0.1×106).
Seven days after leukemia cell injection, mice received 2 intratibial injections of vehicle in one leg, and DBZ in the other leg, at a dose of 10µl of 2 mM, 4 days apart. In other experiments, DBZ was applied i.p. daily thrice starting on day 4 after cell transfer. LSK homing was then performed on day 8. For treating human T-ALL engrafted NSG mice, DBZ was given i.p. starting day 12 after mice receiving 1.6–2.0×106 leukemia cells. Treatment continues for 3 consecutive days out of a 7-day-cycle, at a dose of 10 mg/kg body weight, and repeated up to 4 cycles.
Study of human marrow specimens was approved by the Institutional Research Board (IRB) of the University Hospitals Case Medical Center. Included in the study are 10 control bone marrow with patient age range from 1–17 years (medium age=9.5), 2 T-ALL (age 3 and 27), and 8 B-ALL specimens from patients with age range from 2–23 years (medium age=4.5). See details in supplemental information.
The human T cell leukemia cell lines DND-41 and KOPT-K1 were a gift of Dr. Warren Pear, and were re-tested by sequencing and GSI response in vitro. Cells were plated at 0.5–2×106/ml and cultured in RPMI1640 containing 10% FCS at 37°C with 5% CO2.
Data are presented as means ±SD, unless otherwise stated. Statistical significance was assessed by Student t test or ANNOVA analysis. For survival curve, p values were generated using Mantel-Cox test.
To study the biological influence of ALL leukemia cells on host hematopoiesis, we co-injected retroviral transfected progenitor-enriched marrow cells (Ly5.2) expressing either pMIG-ICN1-eGFP or the control plasmid (pMIG-eGFP), with WT marrow cells (Ly5.1) into lethally irradiated WT hosts (Ly5.1) (Fig 1A). While mice receiving control plasmids (referred hereafter as pMIG-mice) showed no sign of disease, mice receiving ICN1-eGFP developed T-ALL within 4–12 weeks (referred hereafter as ICN1-mice), characterized by the expansion of an immature CD4+/CD8+ T progenitors in the marrow (Fig 1B), thymus and spleen (not shown). Full-blown leukemia was accompanied with a progressive alteration of the hematopoiesis in the non-transformed GFP−Ly5.1+ compartment, characterized by a suppressed B220+ population and an elevation of Gr-1+ cells (Fig 1B; percentage and absolute numbers in Fig1C and 1D, respectively). Hematocrit was not changed (Fig S1A) while platelet number was decreased in mice developing full-blown leukemia (Fig 1E). The GFP−Ly5.1+ marrow also showed an initial expansion of Lin−Sca-1+c-kit+ (LSK) at the earlier stage of reconstitution in both ICN1-mice and pMIG-mice (0.27% and 0.21%, respectively, compared to 0.15% of WT LSK). At the later stage of disease, however, LSK cells were only present at 0.06±0.02% in ICN1-mice but maintained at 0.22±0.04 % in pMIG-mice (Fig 1F, Fig 1G GFP− panel). The frequency and number of megakaryocyte progenitor (MP; c-Kit+CD41+) were reduced (Fig S1B) (25), while the frequency and number of megakaryocyte/erythrocyte progenitor (MEP) and erythroid progenitor (EP) showed a trend of decrease in ICN1-mice (Fig S1C–D). We did not observe altered maturation (data not shown) or increased apoptosis of megakaryocytes isolated from ICN1 mice (Fig S1E). Consistent with reports by others, we also observed a suppression of LSKs in transformed hematopoietic compartment (Fig 1G, GFP+ panel) (26). These findings indicate that lymphoblastic leukemia cells induce a disease stage-dependent reduction of HSPC (LSK and MP) and thrombocytopenia, a mild anemia, a prominent inhibition of B lymphopoiesis but an expansion of granulopoiesis.
To differentiate if inhibition of HSPC and B lymphopoiesis is a result of direct contact-mediated suppression by leukemia cells, we performed in vitro OP9 co-culture, in which either WT lineage-depleted bone marrow (Lin−) or LSKs were co-cultured with leukemia or control cells on OP9 cells (Fig 2A) in the presence of SCF, IL7 and Flt3L (24). In co-culture with Lin− cells, either pMIG-mice marrow cells or WT thymocytes representing immature T cells were used as controls. We found that after 8 days of co-culture, B cell production from Lin− progenitors co-cultured with leukemia cells was significantly reduced compared to those from co-culture with pMIG cells (98.7±6.9 ×103 vs 157.2± 18.2 ×103, p<0.05) (Fig 2B), but not from co-culture with WT thymocytes, suggesting that B cell inhibition was caused by immature T cells rather than leukemia cells. We then examined whether co-culture with leukemia cells could affect the expansion and differentiation of more primitive LSK cells. To avoid excessive LSK differentiation, LSK cells were co-cultured on OP9 with either WT marrow cells (LSK/control), pMIG (LSK/pMIG) or ICN1 (LSK/ICN1) marrow cells for only 4 days (Fig 2A). We found that total numbers of expanded cells (Fig 2C) and expanded progenitors (Lin−Sca-1+) were similar among three culture conditions (Fig 2D). Although there was a mild reduction of B220+ cells (31.5±8.1% and 21±2.8%, p=0.05), and an increase of Gr-1+ cells generated from LSKs co-cultured with ICN1 cells (25.8±9.6% and 47.0±6.4%, p<0.05) when comparing LSK/control with LSK/ICN1 conditions, there was no significant difference in any cell types generated when comparing LSK/ICN1 to LSK/pMIG condition (Fig 2D). In addition, there was no apparent apoptotic alteration in the emerging B220+ cells (data not shown). These results indicate that direct contact with leukemia cells mildly interferes with progenitor cell differentiation along the B and myeloid lineages, but has no obvious effect on the expansion of the progenitor cells.
A mild effect by direct leukemia contact suggests that other mechanism such as changes in the leukemic microenvironment may play a more significant role in hematopoietic suppression. To explore this possibility, we first examined the homing and niche localization of ICN1-transformed pre-leukemia cells using 2-photon imaging analysis. Twenty-four hours after transfer of ICN1- or pMIG-expressing cells into lethally irradiated WT mice, we were able to locate individual GFP+ cells in the calvarium and long bone (data not shown) marrow. Most pMIG-expressing cells were positioned >5 µm from vessel walls (Fig 3A, top), whereas all of the ICN1-expressing GFP+ cells were found either directly attached to or within 5 µm of vessels (Fig 3A, bottom; Fig 3B). On day 8 after cell transfer, duplex and clusters of ICN1-expressing cells were found at peri-vascular regions (Fig 3C, bottom), whereas pMIG-expressing cells were seen throughout the marrow without significant clustering (Fig 3C, top). These ICN1-expressing cell clusters represent proliferating pre-leukemia cells after homing to the marrow as they were not seen on day 1 (Fig 3D). These cells continued to accumulate at peri-vascular regions (Fig 3E, bottom) and populated the entire marrow space in full-blown leukemia mice (Fig 3F), while pMIG-expressing cells were seen mainly as isolated cells (Fig 3E, top).
Next, to examine the effect of peri-vascular accumulation of pre-leukemia cells on normal HSPC homing and niche locations, we co-transferred WT LSK cells with ICN1-expressing or pMIG-cells. When co-transferred with ICN1-expressing pre-leukemia cells, WT LSK cells were found located more distal from the peri-vascular regions (Fig 3G; Fig 3I, right) but closer to the endosteum surface than when co-transferred with pMIG-cells (Fig 3H; Fig 3I, left). Numbers of WT LSK homing to the marrow was decreased by 79% when co-transferred with ICN1-expressing cells (n=15) than with control cells (n=72) (Fig 3G–H). Further, Ki67 index of LSKs was decreased in the presence of ICN-cells (Fig 3J). These findings indicate that leukemia cells out-compete normal HSPC in the peri-vascular distribution, and suppress normal HSPC proliferation.
To examine cellular alterations mediated by leukemia cells, we quantified numbers of marrow stromal cells including osteoblastic lineage cells (OB) and multi-potent stroma cells (MSC) (Fig 4A) in age-matched pMIG-mice (top) and ICN1-mice (bottom). Consistent with histologic examination showing decreases of OBs in ICN1-mice (Fig 4B), we found a remarkable decrease of OBs in ICN1-mice (Fig 4C), while MSC (Fig 4D) numbers were variable. Isolated OBs from pMIG- and ICN1-mice were then examined for their RNA expression of osteoblast transcription factors. Indeed, we found that RNA levels of Runx2, osterix, osteocalcin, ostegrin, and CXCL12 were all decreased in ICN1-mice OBs (Fig 4E), while overall stroma cells of ICN1-mice (by selecting CD45−TER119−CD31− population) showed increased expression of IL-6, SCF, HIF1α, VEGFα, and Notch ligand Jagged1 (JAG1) (Fig 4F).
Because JAG1 RNA expression was up-regulated in leukemia marrow, we examined whether Notch activation was indeed responsible for the observed phenotype in ICN1-mice. We found that Notch1 and Notch2 receptor as well as several Notch target expressions in ICN1-mice OBs and MSCs were all increased compared to those of pMIG-mice (Fig 5A). Possible contamination by leukemia cells was excluded (Fig S2). We then co-cultured osteoblastic progenitors, MC3T3, with pMIG- or ICN1-cells (Fig 5B). We confirmed that JAG1 protein expression was elevated in leukemia stroma (not shown) as well as in MC3T3 cells exposed to ICN1-cells (Fig 5C). Further, we observed an ICN1 induced up-regulation of Notch1, Hes1 and Hey1 in MC3T3 cells (Fig 5D), while co-culture of MC3T3 with ICN-cells in the presence of GSI, dibenzazepine (DBZ), completely reversed the induction of Notch1 and Hes1 expression (Fig 5C–D). To investigate if exposure to leukemia cells interferes with osteoblast hematopoietic supportive function, we first pre-treated MC3T3 cells with leukemia cells or controls for 2 days, and then co-cultured MC3T3 with WT LSKs (Ly5.2) for 4 days after removal of leukemia or control cells (Fig 5E). Expanded LSK cells (Fig 5F) were then co-transplanted with WT marrow cells (Ly5.1) to lethally irradiated mice. Three months after transplantation, cells (Ly5.2) derived from ICN1-pretreatment culture displayed similar chimerism and proliferation as cells from control-treated culture (not shown), but showed a modestly skewed myeloid differentiation at the expense of B lymphocytes (Fig 5G). In combined, these findings are reminiscent of de novo leukemia mice showing suppression of B lymphopoiesis and expansion of granulocytes. However, we suspect that a more prominent HSPC suppression requires either other stromal components, or more likely, a sustained in vivo leukemia milieu.
Because CXCL12-expressing stroma cells are essential components of the stem cell niche (27, 28), we then investigated whether Notch activation in osteoblasts inhibits CXCL12 expression (Fig 4E). Notch regulates gene expression by forming transcriptional complex with the DNA binding protein RBPJ/CSL. We found that ~1.1kb upstream of CXCL12 promoter contains the RBPJ/CSL sequence (TGGGAA) (Fig S3A) (29), and confirmed this by CHIP analysis (Fig S3B). In luciferase reporter assay, co-transfection of the CXCL12 promoter construct including the RBPJ/CSL binding sequence with RBPJ siRNA into MC3T3 resulted in ~2.5-fold increase of luciferase activity (Fig 5H), whereas its co-transfection with either activated Notch1 (ICN1) or Notch2 (ICN2) reduced activity by 60% and 53%, respectively (Fig 5I–J). These findings suggest that Notch activation in osteoblasts negatively regulates CXCL12 expression. However, the mechanism whereby a transcriptional activator formed by Notch/RBPJ negatively regulates CXCL12 remains unclear and warrants further investigation.
We then tested whether blocking Notch activation could rescue the suppression of osteoblast and hematopoiesis. Non-irradiated WT recipients receiving leukemia cells from primary ICN1-mice readily develop secondary leukemia within 2 weeks (26). Mice then received intratibial injection of GSI (DBZ), or vehicle, ipsilaterally. Analysis of tibia morrow 1 week later revealed that, compared to vehicle-treated marrow, DBZ-treated marrow displayed a partial restoration of OB (Fig 6A–B) and LSK cells (Fig 6C–D), and modestly increased MP numbers (Fig S4A). DBZ treatment of pMIG-mice (control) showed no significant effect. DBZ treatment also modestly corrected expanded Gr-1 and suppressed B220 cells (Fig 6E) in leukemia marrow but had no obvious effect in the control marrow. These effects mediated by DBZ are unlikely results of leukemia burden reduction, as cleaved ICN1 cells are not direct target of GSI; and indeed, we do not see a significant reduction of GFP+ cells in DBZ-treated tibia (35±8.2%) compared to vehicle-treated tibia (39±5.6%). We then treated secondary leukemia mice systemically with DBZ. Compared to control-treated mice that had 100% mortality by day 15 (day 11–14 peripheral GFP+ level: 37.8±6.5%), DBZ treatment of ICN1-mice (day 11–14 peripheral GFP+ level: 34.2±8.1%) improved survival such that 40% of mice lived longer than 20 days (Fig 6F; p<0.05). Homing of WT LSK cells to DBZ-treated leukemia marrow also improved by 40% (Fig 6G), with more LSK cells identified in the vicinity of peri-vascular region in DBZ-treated marrow (Fig S4B, bottom) compared to non-treated marrow (Fig S4B, top). In addition, DBZ treatment increased ki67 index of engrafted LSKs (Fig 6H) and osteoblast cell-surface expression of CXCL12 in leukemia mice (Fig 6I–J). Further, systemic Notch blockade suppressed elevated expression of IL-6 and some of Notch targets, but reversed stromal expression of CXCL12, Runx2, and osterix (Fig 6K). Systemic Notch blockade also improved platelet numbers in secondary leukemia mice (Fig S4C); however, it had no significant effect on hematocrit or MEP (Fig S4D–E). In summary, these findings suggest that leukemia-mediated osteoblastic alteration and suppression of marrow niche function are substantially improved by blocking Notch activation.
To examine if human T-ALL mediate similar hematopoietic and stromal suppression, we engrafted NSG mice with human T-ALL cells (14). We found that NSG mice engrafted with DND41 (Fig 7A) or KOPT-K1 (data not shown) showed suppression of non-human LSK populations that was partially reversed by DBZ treatment. Both OB and MSC frequencies (Fig 7B) and their cell surface expressions of CXCL12 (Fig 7C) were decreased in leukemia developing mice but were much improved in mice receiving DBZ treatment. Further, non-hematopoietic stromal cells in DND41-engrafted NSG mice displayed down-regulation of Runx2 and CXCL12, and up-regulation of Deltex, JAG1 and Hey2 (Fig 7D) that were reversed by DBZ treatment. Therefore human T-ALL cells also show GSI-reversible OB and HSPC suppression as mouse T-ALL cells, although such responses are likely also contributed by decreased leukemia burden following treatment (84±8% in non-treated mice vs 65±10% in treated mice, p< 0.05) since these T-ALL cells still retain γ-secretase cleavage sites (14).
To further examine the relevance of our findings in human ALL, we examined sampled (n=10) non-leukemic human bone marrow sections and age-matched ALL marrow sections from 2 T-ALL and 8 B-ALL patients. Bone-lining cuboid shaped or flattened osteoblasts are readily identified in non-leukemic bones (Fig 7E), but are notably decreased in 2 T-ALL (Fig 7F) specimens, and are also modestly decreased in B-ALL bone marrow (Fig 7G). In addition, compared to 2 non-leukemia tissues (Fig 7H), Hes1 expression was increased showing stronger nuclear staining in the remaining osteoblasts in 2 T-ALL (Fig 7I) and also in 6 of 8 B-ALL specimens. These findings of aberrant Notch activation in osteoblasts and a general loss of osteoblasts are in agreement with findings from animal models and human T-ALL grafts.
Proliferation of leukemia cells in acute leukemia patients is often associated with anemia and cytopenia. This condition is generally believed to reflect a “crowding-out” effect by the rapidly proliferating leukemia cells. Emerging evidences, however, indicate that leukemia cells modulate the marrow microenvironment to disrupt the communication between the stem cell niche and the residing HSPCs that is essential for hematopoietic homeostasis. In this study, using a mouse model of T-ALL, we found that lymphoblast leukemia cells induce a marked suppression of normal hematopoiesis by harnessing two critical niche components, the osteoblastic lineage cells and the peri-vascular region. We identified that aberrant Notch activation plays an important role in these processes since blocking Notch activation can largely recovers suppressed osteoblast numbers and increases HSPC proliferation, and improves animal survival as well as thrombocytopenia. We show that, at least in ICN1 T-ALL mouse model, an improved hematopoiesis and survival is a direct result of leukemia microenvironment change rather than a consequence of decreased leukemia burden. Notably, we confirmed that osteoblast loss and associated Notch activation were present in human T-ALL xenografts as well as in human ALL specimens.
Osteoblasts are defined as MSC progeny committed to the osteoblastic lineage expressing many cell-signaling molecules such as JAG1, CXCL12, membrane-bound SCF, angiopoietin-1, osteocalcin, and osteopontin, some of which are critical to support HSC self-renewal and survival (9, 23, 30). Mature osteoblasts also directly regulate B lymphopoiesis (31, 32). Recent studies have linked dysfunctional osteoblast expansion with disease pathophysiology in acute and chronic myeloid leukemia (33, 34). However, the significance of osteoblast in modulating lymphoblastic leukemia microenvironment has not been studied. Here we show that unlike in myeloproliferative neoplasia, osteoblast population is strikingly suppressed in mice developing T-ALL. We also show that osteoblast suppression is associated with a loss of normal HSPC population, and suppressed B lymphocytes, consistent with the dual function of these cells in supporting HSPC homeostasis and B lymphopoiesis. Further, we identified a critical role of Notch activation in osteoblast suppression. The implication of Notch as the major pathway to suppress osteoblast differentiation is not surprising as Notch activation has been shown to inhibit osteoblastic progenitor terminal differentiation through Hes1 repressing Runx2 transcriptional activity (35–37). Our findings further suggest that osteoblast CXCL12 expression is negatively regulated by Notch activation. Finally, variable degree of osteoblast loss is observed in human T-ALL as well as B-ALL specimens. Although we show that blocking Notch activation could partially account for the recovery of dysregulated osteoblastic niche functions, neither have we excluded contributions by other pathways such as Wnt or TGF-β in the suppression of HSPC, nor have we examined other molecular mechanisms contributing to disease-specific suppression of osteoblasts and their niche function in various forms of human ALL.
Besides osteoblasts, peri-vascular niche and related cells are another major focus of research in leukemia microenvironment. Peri-vascular homing provides leukemia cells proliferative and survival cues, and also promotes dysregulated angiogenesis in the marrow, linking marrow to be a common site of relapse for hematologic malignancies such as T-ALL (38, 39). Marrow could serve as sanctuary sites for the integration of leukemia cells and contributes to the leukemic potential of transformed endothelial cells (40). In a pre-B ALL xenograft model, CXCL12/CXCR4 axis signaling was found to support leukemia metastasis to the specialized micro-vascular domain (5). Consistent with other reports (41, 42), CXCR4 expression was found increased in leukemic T cells. However, GSI did not inhibit increased CXCR4 expression nor affect leukemia homing to the peri-vascular region (data not shown), indicating that either Notch activation is not responsible for the increased CXCR4 expression and preferred peri-vascular homing of leukemia cells, or that other pathways or molecules also regulate CXCR4 expression. Nevertheless, lymphoblastic leukemia cells out-compete normal HSPC and displace HSPC from the peri-vascular region, and contributes to the suppression of HSPC proliferation. In addition, disruption of endothelial niche where megakaryocytes are normally in close interaction with HSC may explain platelet abnormality observed in ICN1-mice (43). Although MEP and EP were mildly decreased in ICN1 leukemia marrow, prominent anemia seen in acute leukemia patients was not found in these leukemia mice. It is possible that severe anemia may take longer to develop in mice. Alternatively, splenomegaly-associated increase of EPs and extramedullary hematopoiesis could compensate for the reduction of marrow red cell production (data not shown).
Leukemia-mediated environmental change also implicates abnormal angiogenesis (44), hypoxia (45), or the inflammatory cytokines such as SCF and IL-6 (33) (46). Increased inflammatory cytokines were found responsible for supporting reinforced leukemia-niche for CML cells (23). Notably, inflammatory cytokines including IL-6 was found increased in the marrow stroma of T-ALL driven by ICN1. Increased IL-6 is associated with poor response to GSI in cancer cell lines (47). We found that IL-6 was suppressed after GSI treatment, suggesting that alterations of inflammatory cytokines are likely a secondary effect downstream of Notch activation in T-ALL. Stroma Notch activation could be induced by up-regulated Notch ligand (Fig 5C), hypoxia, or inflammation. Further studies are required to elucidate the exact molecular mechanism underlying stromal NOTCH activation in ALL leukemic marrow, and the molecular links between aberrant Notch activation and inflammatory cytokine increase implicated in leukemia progression and therapy resistance.
In summary, our findings reveal that during disease progression, lymphoblastic leukemia employs disease-specific cellular and molecular machinery to dysregulate hematopoietic niche function. It is known that in relapsed ALL, re-induction with aggressive chemotherapy or stem cell transplantation is associated with long-term sequelae and overall poor outcomes (48). Knowledge gained from our studies thus provides rationale of developing niche-targeted approach in the future to improve clinical outcome in ALL patients.
This work was supported by grants from American Cancer Society LIB-125064 (L.Z.), NIH HL103827 (L.Z.), Hyundai Hope-on-Wheels Program (A.Y.H), and the Keira Kilbane Cancer Innovation Fund (A.Y.H).
Disclosure of potential conflicts of interest: The authors declare no competing financial interests related to this work