Leukemic Stem Cells In Bone Marrow Research Articles

Nanoparticle-Mediated Delivery of Micheliolide Analogs to Eliminate Leukemic Stem Cells in the Bone Marrow.

Micheliolide (MCL) is a naturally occurring sesquiterpene lactone that selectively targets leukemic stem cells (LSCs), which persist after conventional chemotherapy for myeloid leukemias, leading to disease relapse. To overcome modest MCL cytotoxicity, analogs with ≈two-threefold greater cytotoxicity against LSCs are synthesized via late-stage chemoenzymatic C-H functionalization. To enhance bone marrow delivery, MCL analogs are entrapped within bone-targeted polymeric nanoparticles (NPs). Robust drug loading capacities of up to 20% (mg drug mg-1 NP) are obtained, with release dominated by analog hydrophobicity. NPs loaded with a hydrolytically stable analog are tested in a leukemic mouse model. Median survival improved by 13% and bone marrow LSCs are decreased 34-fold following NPMCL treatments versus controls. Additionally, selective leukemic cell and LSC cytotoxicity of the treatment versus normal hematopoietic cells is observed. Overall, these studies demonstrate that MCL-based antileukemic agents combined with bone-targeted NPs offer a promising strategy for eradicating LSCs.

Leukemic stem cells shall be searched in the bone marrow before "tyrosine kinase inhibitor-discontinuation" in chronic myeloid leukemia.

Leukemic stem cells (LSCs) of chronic myeloid leukemia (CML), persisting in the bone marrow (BM) niche, could be responsible for the relapses within the patients of whom the treatment-free remission (TFR) had been attempted. We assessed the presence of the CML LSCs in the peripheral blood (PB) and concurrently in the BM in the patients with chronic-phase CML (CP CML). Thirty-eight patients with CP CML were included into the study. CD45+ /CD34+ /CD38- cells with positive CD26 expression were considered as CML LSCs (CD26+ LSC) by using multiparameter flow cytometry (FCM). Mean BCR-ABL, PB LSC, and BM LSC were 58.528 IS (37.405-83.414 IS), 237.5LSC/μL (16-737.5LSC/μL), and 805LSC/106 WBCs (134.6-2470LSC/106 WBCs), respectively, in newly diagnosed CML patients. In the patients with BCR-ABL positive hematopoiesis, mean BCR-ABL, PB LSCs, and BM LSCs were 30.09IS (0.024-147.690IS), 13.5LSC/μL (0-248.7LSC/μL) and 143.5LSC/106 WBCs (9-455.2LSC/106 WBCs), respectively. No CML LSCs were detected in PB of patients who achieved deep molecular response (DMR). BM LSCs of the patients who were in DMR were 281.1LSC/106 WBCs (3.1-613.7LSC/106 WBCs). The amount of PB LSCs was highest in patients with newly diagnosed CML (P<.001). LSCs persisted in the BM of the patients with DMR, whereas there was no LSCs in the peripheral blood. The investigation of the CML LSCs in bone marrow before deciding TKI discontinuation could be justified to achieve and maintain stable TFR.

FLT4 Expressing CD34+CD38- Leukemic Stem Cells in Bone Marrow of Refractory Patients are Protected by Receptor Internalization under Abundant VEGF-C Condition

So far, the cause of relapse or refractoriness in acute myeloid leukemia (AML) is regarded as the persistence of chemoresistant leukemic stem cells (LSCs). Even VEGF-C/FLT4 axis mediates blast proliferation and survival, understanding of the dynamic behavior of LSCs in bone marrow remains elusive. Especially, FLT4 endocytosis by VEGF-C is related to various biologic functions including signal activation. Based on our previous study, which addressed the potential of FLT4 as a marker for LSCs, we showed that surface FLT4 can be internalized by VEGF-C exposure in bone marrow (BM) derived LSCs. It resulted in resistant to chemotherapy, suggesting LSCs protection. In addition, data for apoptosis revealed that inhibition of FLT4 using MAZ51 with cytosine arabinoside (Ara-C) can effectually increase cell death under abundant VEGF-C in refractory patients. It indicated that targeting FLT4 allows investigators to establish advanced therapeutic strategy with conventional Ara-C in refractory patients with high VEGF-C. In clinic, low level of surface FLT4 in de novo AML-BM LSCs, but not PB cells were inclined to undergo refractory status after induction chemotherapy. To investigate whether FLT4 expressing CD34+CD38- LSCs can be protected by VEGF-C via internalization, FACS analysis, western blotting and immunocytochemistry were performed. Meanwhile, total 66 newly diagnosed (ND) AML patients were used to analyze for clinical correlation. (BM, n=37; PB, n=19). FACS analysis showed that surface FLT4 expressing CD34+CD38- LSCs in BM, but not PB, were significantly decreased by VEGF-C (In BM; without VEGF-C & with VEGF-C, 38.7±8.1% & 16.0±5.1%). Similar with surface FLT4 in PB cells, both intracellular FLT4 in BM and PB was highly sustained, regardless of VEGF-C (In BM; without VEGF-C & with VEGF-C, 62.9±17.2% & 69.7±14.9%, In PB; without VEGF-C & with VEGF-C, 77.4±15.6% & 70.4±21.8%). Immunocytochemistry and western blotting also showed internalization of surface FLT4 by VEGF-C treatment. FLT4+CD34+CD38- cells both in BM and PB were significantly higher in refractory patients than in post allogeneic stem cell transplantation (SCT) group, implying clinical correlation of FLT4 with refractory patients. Consistent with findings in ND-AML and refractory groups, FLT4+CD34+CD38- cells were highly sustained in complete remission group, showing drug resistant population (ND-AML, n=30; refractory AML, n=9; Complete remission AML, n=18; post-allogeneic stem cell transplantation, n=9). High level of VEGF-C was detected in refractory patients, compared to that of normal donors (Refractory AML, n=9, normal donors, n=7). Apoptosis results showed that high number of apoptotic CD45dimCD34+CD38- cells in MAZ51 (FLT4 antagonist) and Ara-C dual treatment under VEGF-C exposure, compared to no treatment of both MAZ51 and Ara-C (without VEGF-C; 0.5-1.3 folds, with VEGF-C; 2.7-43.3 folds) or single exposure either to MAZ51 or Ara-C (In Ara-C only; without VEGF-C; 1.0-9.5 folds, with VEGF-C; 0.1- 1.3 folds, In MAZ51 only; without VEGF-C; 1.1-1.7 folds, with VEGF-C; 1.3-39.3 folds), suggesting that Ara-C induced-blast apoptosis can be augmented by FLT4 inhibition even in the presence of VEGF-C in a sub-group of either refractory or relapsed AML patients. VEGF-C/FLT4 axis in AML is involved in PI3K and AKT pathway.Collectively, we demonstrated that FLT4 internalization under VEGF-C in BM leads to protection of FLT4 expressing LSCs and is clinically relevant to refractory subgroup. This data could suggest some clues to develop therapeutic strategies in targeting FLT4 expressing refractory LSCs. DisclosuresNo relevant conflicts of interest to declare.

Early and Late Leukemic Stem Cells in Murine Leukemia Models

Acute leukemia is uncontrolled proliferation of leukemic stem cells (LSCs). Murine models of leukemia suggest that LSCs arise from lineage-committed progenitor cells. However, whether LSCs also directly arise from long-term (LT) and short-term (ST) hematopoietic stem cells (HSCs) and other hematopoietic progenitor cells (HPCs) and whether differentiation potentials influence the leukemia types are poorly understood.In this study, we used two murine leukemia models (AML with MLL-AF9 fusion protein and T-ALL with Notch-1 intracellular domain, ICN-1). Two HSC and three HPC populations were sorted from B6 (CD45.1) mouse bone marrow (BM) by flow cytometry: HSC1, CD150+CD41-CD34-Lin-Sca-1+c-Kit+ (LSK) cells; HSC2, CD150-CD41-CD34-LSK cells; HPC1, CD150+CD41+CD34-LSK cells; HPC2, CD150+CD41+CD34+LSK cells; HPC3, CD150-CD41-CD34+LSK cells. HSC1, HSC2, HPC1, and HPC3 are enriched in LT-HSCs, ST-HSCs, repopulating common myeloid progenitors, and lymphoid-primed multipotent progenitors, respectively. 400-600 cells were sorted from each population, pre-stimulated for 24 hrs, and transduced with MLL-AF9 or ICN-1 retrovirus for another 24 hrs. Cells were mixed with 3-5 x 105 BM cells (CD45.2), and injected into the lethally irradiated mice (CD45.2). The recipient mice were monitored by detection of GFP+ cells in the peripheral blood (PB). When the recipient mice showed > 50% GFP+ cells in the PB or became ill, mice were killed and analyzed for leukemia.In the MLL-AF9 AML model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 6 weeks after transplantation. Leukemic cells in PB and BM appeared positive for Mac-1/Gr-1, but negative for CD3 and B220. We detected a new LSC population in the BM: Lin-Mac-1+c-Kit+Sca-1+CD150-CD16/32+ cells. Both CD34-negative and CD34-positive cells were detected in this population. We named this population as "early LSCs" because it was phenotypically similar to ST-HSCs and/or LMPPs in normal mouse BM cells except Mac-1 expression. Early LSCs differed from previously identified LSCs (L-GMP) because they are Sca-1-positive. We now consider L-GMP as one of "late LSC" types. Early LSCs formed leukemic colonies in vitro and initiated the AML in serial transplantation. These results show the similar AML develops regardless of HSC and HPC types transduced. Both early and late LSCs likely play a role in establishment of AML.In the ICN-1 T-ALL model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 4 weeks after transplantation. Most leukemic cells in PB and BM appeared positive for CD4/CD8, but negative for Mac-1/Gr-1 and B220. We did not detect early LSCs in BM. To identify late LSCs in BM, we injected the CD3+CD8+CD4- and CD3+CD8+CD4+ cells into the sub-lethally irradiated mice. T-ALL developed from both populations (median latency, 23 vs 35 days). These results show the similar T-ALL develops regardless of HSC and HPC types transduced. In this case, late LSCs can be directly generated without early LSCs from HSC or HPC populations.Taken together, we found that the similar types of leukemia develop regardless of different types of initiating cells in both models. We also found new LSC populations, early LSCs in the AML model and late LSCs in the T-ALL model. Both early and late LSCs were able to re-initiate leukemia after secondary transplantation.In conclusion, highly purified murine HSCs and HPCs were used for the first time to initiate leukemia. Both MLL-AF9 AML and ICN-1 T-ALL models suggest that gene mutations at all differentiation stages from HSCs to HPCs potentially induce the similar types of leukemia. Of note is that distinct lineage differentiation potentials of HSCs and HPCs do not affect leukemia types. More importantly, early LSCs may serve as the earliest event in leukemogenesis in AML. In the case of ALL, gene mutations seem carried over until reaching the developmental stage of late LSCs. Our results suggest that both early and late LSCs should be eradicated to achieve complete remission and prevent relapse. DisclosuresNo relevant conflicts of interest to declare.

DYRK2 Inhibits the Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Inducing Degradation of c-Myc Downstream of the Reprogramming Factor KLF4

Chronic myeloid leukemia (CML) is a blood cancer originated by expression of BCR-ABL, a constitutively activated kinase product of the chromosomal translocation t(9;22), in hematopoietic stem cells (HSC). Although tyrosine kinase inhibitors (TKI) can efficiently induce molecular remission in CML patients, drug discontinuation often leads to relapse caused by reactivation of leukemic stem cells (LSC) spared from TKI therapy via BCR-ABL-independent mechanisms of self-renewal and survival. Thus, there is a need for alternative drugs for relapse patients to prevent expansion of BCR-ABL-positive LSC during discontinuation of chemotherapy or emergence of chemoresistance. We found that somatic deletion of the reprogramming factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by a progressive attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. Analyses of global gene expression and genome-wide binding of KLF4 revealed that the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) is repressed by KLF4 in CML LSCs. Immunoblots revealed elevated levels of DYRK2 protein that were associated with a reduction of c-Myc protein and increased levels of p53 (S46) phosphorylation and PARP cleavage in KLF4-deficient LSCs purified from the bone marrow of CML mice. Genomic silencing of KLF4 in the murine CML cell line 32D-BCR-ABL resulted in increased levels of DYRK2 and phosphorylated c-Myc (S62) leading to diminished levels of c-Myc protein, which was reverted by treatment with a proteasome inhibitor, suggesting that KLF4 prevents c-Myc degradation triggered by DYRK2-mediated priming phosphorylation. Consistent with an inhibitory role in leukemia, DYRK2 levels are significantly reduced both in CD34+CD38+ and CD34+CD38− cells from CML patients compared to normal stem/progenitor cells. Aiming at pharmacological activation of DYRK2 to abrogate self-renewal and survival of CML cells, we treated CML cells with vitamin K3 that inhibits Siah2, an ubiquitin E3 ligase involved in Dyrk2 proteolysis. Vitamin K3, and not Vitamin K1 and K2, induces dose-dependent cytotoxicity in a panel of human-derived CML cell lines by stabilizing Dyrk2 protein and consequently promoting c-Myc degradation. Interestingly, combination of vitamin K3 with Imatinib exhibit additive effect inducing cytotoxicity in CML cells. Collectively, the identification of Dyrk2 as a critical mediator of LSC downfall is a novel paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in conjunction with Imitinib in CML patients. DisclosuresNo relevant conflicts of interest to declare.

Vcam1 Is a "Don't-Eat-Me" Signal on Healthy Hematopoietic and Leukemic Stem Cells

▪Hematopoietic stem cells (HSCs) possess the ability to maintain the entire population of blood cells throughout life and to replenish the hematopoietic system after transplantation into marrow-ablated recipients. In mammalian adults, HSCs predominantly reside in the bone marrow to generate and maintain all blood cell types. HSCs, however, retain their ability to migrate to ectopic niches via the bloodstream, and traffic back to the bone marrow microenvironment via in part the interactions of alpha4 integrins with Vascular Cell Adhesion Molecule-1 (Vcam1, also known as CD106), constitutively expressed by endothelial and stromal cells. In the course of studies to investigate the role of Vcam1 in the macrophage erythroblastic niche, we have found using Vcam1-floxed mice crossed with a Csf1r-iCre (hereafter Vcam1Δ/Δ) that Vcam1 and Cre, using this transgenic line, were expressed on Lineage− (Lin−) c-Kit+ Sca1+ CD48− CD150+ HSCs. Although Vcam1Δ/Δ mice show no significant steady-state hematopoietic defect, transplantation of Vcam1Δ/Δ bone marrow, both in competitive and non-competitive settings, failed to engraft irradiated wild-type recipients. Indeed, ~80% of animals transplanted with Vcam1Δ/Δ cells died, and the few that survived failed to successfully engraft secondary recipient mice. Vcam1Δ/Δ HSC/progenitors homed to bone marrow as well as wild-type counterparts and showed no detectable change in apoptosis in the bone marrow. However, syngeneic Vcam1Δ/Δ HSC/progenitors were selectively cleared in vivo by bone marrow macrophages over 4 days. The in vivo Vcam1Δ/Δ HSC/progenitor clearance did not require irradiation-induced damage since in steady-state parabiosis (Vcam1Δ/Δ and wild-type co-joined pairs), Vcam1Δ/Δ HSCs did not engraft the partner after G-CSF-induced mobilization whereas wild-type HSCs engrafted the parabiont partner. In addition, in vitro incubation of bone marrow-derived macrophages with Lin− cells revealed rapid phagocytosis by macrophages of Vcam1Δ/Δ, but not wild-type, cells and phagocytosis was also induced when the integrin alpha4 beta1 (also known as VLA-4) was blocked in wild-type Lin− cells. Thus, these results suggest that Vcam1 expressed on HSCs serves as a novel don’t-eat-me signal, controlling the migration in the bone marrow by a macrophage-enabled checkpoint. Since we found that Vcam1 is expressed at higher levels on acute myelogenous leukemia (AML) cells than on healthy HSCs, and high VCAM1 expression correlates with poor prognosis in human AML patients (Ley et. al. NEJM 2013), we examined whether Vcam1 expression by AML cell could affect leukemogenesis in vivo. To test this idea, we generated AML-Vcam1Δ/Δ and AML-Control cells by transduction of bone marrow Lin− c-Kit+ Sca1+ cells with the pMSCV-MLL-AF9-GFP oncogene. Strikingly, FACS analysis of primary AML recipient bone marrow revealed a marked reduction (>99%) in the number and percentage of phenotypic Lin− IL7Rα− Sca1− MLL-AF9 GFP+ c-Kithigh CD34low FcγRII/IIIhigh leukemic stem cells in AML-Vcam1Δ/Δ mice compared to control. These results were confirmed by imaging analyses, which showed a marked reduction in leukemic infiltration in AML-Vcam1Δ/Δ mice compared to control. Kaplan-Meier survival analysis of secondary recipient mice receiving 20,000 GFP+ leukemic cells revealed a significantly greater survival of mice harboring AML-Vcam1Δ/Δ cells relative to AML-Control (P<0.0001). To assess in a pre-clinical experimental setting whether Vcam1 inhibition can alter the course of AML, we established AML in immunocompetent C57BL/6 recipients and started therapy of moribund leukemic mice (>50% circulating AML-GFP+ cells) with a daily injection of saline, IgG control, anti-Vcam1 blocking antibody (100 μg/day), cytarabine (100 mg/kg), or a combination of anti-Vcam1/cytarabine for 5 days. Remarkably, this short treatment with anti-Vcam1 blocking antibody was able to preferentially and significantly reduce the frequency and absolute number of phenotypic bone marrow leukemic stem cells in vivo. In addition, our results suggest that using a blocking anti-Vcam1 antibody synergizes with cytarabine treatment to decrease leukemic burden in vivo. These studies lay the groundwork for the development of a new therapeutic strategy for eliminating leukemia stem cells by modulating the innate immune response. DisclosuresFrenette:GSK: Research Funding; Pfizer: Consultancy; PHD Biosciences: Research Funding.

KLF4 Regulates Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Repressing Gbl Expression and Altering mTORC2 Activity

Abstract Tyrosine kinase inhibitors (TKIs) are the standard treatment for eradicating BCR-ABL-positive progenitor cells in chronic myeloid leukemia (CML); however, disease often relapses upon drug discontinuation because TKIs do not effectively eliminate leukemic stem cells (LSC). The development of novel strategies aimed at eradicating LSC without harming normal hematopoietic stem cells (HSC) is essential for the cure of CML patients. The generation of LSC-directed therapy relies on the identification of novel molecular pathways that selectively regulate LSC function independent of BCR-ABL. The Krüppel-like factor 4(KLF4) is a transcription factor that can either activate or repress gene transcription acting as an oncogene or a tumor suppressor depending on the cellular context. Analysis of a published dataset from chronic phase CML patients revealed elevated levels of KLF4 in LSC compared to progenitor cells indicating that KLF4 is likely implicated in LSC regulation. To study the role of KLF4 in LSC function, we used a CML mouse model combining somatic deletion of the Klf4 gene and retroviral transduction and transplantation of HSC. In contrast to mice receiving BCR-ABL-transduced Klf4fl/fl HSC that developed and succumbed to CML, mice transplanted with BCR-ABL-transduced Klf4Δ/Δ (Klf4fl/fl Vav-iCre+) HSC showed a progressive loss of leukemia despite an initial expansion of myeloid leukemic cells, which led to increased overall survival. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSC in bone marrow and the spleen. Furthermore, deletion of KLF4 impaired the ability of LSC to recapitulate leukemia in secondary recipients suggesting a loss of self-renewal capacity. In contrast to LSC, KLF4 deletion led to increased self-renewal of normal HSC assessed by serial competitive transplantation. To identify KLF4 target genes involved in LSC self-renewal, we performed a global gene expression analysis using Klf4Δ/Δ LSC purified by cell sorting from leukemic mice. Analysis of gene expression in Klf4Δ/Δ LSC revealed significant upregulation of GβL, a component of mTOR complexes. Finally, we identified that KLF4 binds to GβL promoter by Chip-Seq analysis and that silencing resulted in inhibition of mTORC2 but not mTORC1 activity in 32D-BCR-ABL-positive CML cells. Our findings suggest that KLF4 transcriptionally represses GβL expression in LSC and that mTORC2 inhibition has the potential to completely eradicate LSC and induce treatment-free remission. Disclosures No relevant conflicts of interest to declare.

Resistance of leukemic stem-like cells in AML cell line KG1a to natural killer cell-mediated cytotoxicity

Leukemic stem cells (LSCs) play the central role in the relapse and refractory of acute myeloid leukemia (AML) and highlight the critical need for the new therapeutic strategies to directly target the LSC population. However, relatively little is known about the unique molecular mechanisms of drug and natural killer cells (NK)-killing resistance of LSCs because of very small number of LSCs in bone marrow. In this study, we investigated whether established leukemia cell line contains LSCs. We showed that KG1a leukemia cell line contained leukemic stem-like cells, which have been phenotypically restricted within the CD34+CD38− fractions. CD34+CD38− cells could generate CD34+CD38+ cells in culture medium and had renewal function. Moreover, CD34+CD38− cells had self-renewal potential. We found that leukemic stem-like cells from KG1a cells were resistant to chemotherapy and NK-mediated cytotoxicity. NKG2D ligands involve in protecting LSCs from NK-mediated attack. Taken together, our studies provide a novel cell model for leukemic stem cells research. Our data also shed light on mechanism of double resistant to chemotherapy and NK cell immunotherapy, which was helpful for developing novel effective strategies for LSCs.

Abstract LB-45: High aldehyde dehydrogenase activity is a marker for normal hematopoietic stem cells but not leukemic stem cells in acute myeloid leukemia: novel therapeutic implications.

Abstract Only a minority of cells, the leukemic stem cells (LSC), within AML are responsible for tumor growth and maintenance. Many patients experience relapse after therapy which originates from outgrowth of therapy resistant LSC. Therefore, eradication of LSC is necessary to cure AML. Both the normal hematopoietic stem cells (HSC) and LSC co-exist in the bone marrow (BM) of AML patients and success of anti-LSC strategies relies on specific elimination of LSC while sparing HSC. LSC are contained within the CD34+CD38-, the side population (SP) and the high aldehyde dehydrogenase (ALDH) activity compartments. ALDH is a detoxifying enzyme responsible for oxidation of intracellular aldehydes and high ALDH activity results in resistance to alkylating agents such as cyclophosphamide. It has been shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blasts. In view of applicability of LSC specific therapies the detoxification by ALDH is clinically very important. A difference in ALDH activity between HSC and LSC might be used to preferentially kill LSC while sparing HSC. To establish ALDH activity differences between HSC and LSC it should be possible to discriminate between them. We have shown that LSC can be identified and discriminated from HSC using stem cell-associated cell surface markers, such as CLL-1, lineage markers (CD7, CD19, CD56) and recently CD34/CD45 expression and cell size characteristics (Terwijn, Blood 111: 487, 2008). This offers the opportunity to identify co-existing LSC and HSC in the AML BM. We now show that, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that HSC that co-exist with LSC in BM of AML patients have a higher ALDH activity as compared to their malignant counterparts. We have analyzed ALDH activity in HSC and LSC, both present in the BM from 18 AML patients. In nine BM AML samples, defined as CD34negative (&amp;lt;1%CD34+ blasts), the CD34+ compartment contained only normal CD34+CD38− HSC. The ALDH activity in these CD34+ HSC, is a factor 4,4 (range 1,7–18,9) higher than in LSC. In nine BM AML samples, defined as CD34positive AML, the CD34+CD38- HSC have a 7,7 fold (range 1,73–29,2 fold) higher ALDH activity as compared to putative LSC. In both CD34-positive and CD34-negative AML, we confirmed the identity of HSC and LSC by screening for molecular aberrancies present in AML blasts. The level of the ALDH activity of HSC within the AML BM is similar to that of HSC in NBM of healthy donors. In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM (irrespective of AML phenotype) at diagnosis. Consequently, AML patients with high ALDH activity in HSC might benefit from treatment with agents that will be converted by ALDH enzymes, such as cyclophosphamide, whereby the difference between the activity in LSC and HSC will define the therapeutic window. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-45. doi:10.1158/1538-7445.AM2011-LB-45

Increase in Leukemic Stem Cells Is Associated with Acceleration of BCR-ABL-Induced CML in P-Selectin-Deficient Mice.

The BCR-ABL oncogene causes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) in human, and CML-like disease and lymphoid leukemia in mice. Recent studies show that cell adhesion molecule P-selectin is a potent negative regulator of human hematopoietic progenitors. Our previous work shows that lack of P-selectin accelerates the development of BCR-ABL-induced CML in mice (Shawn et al, Blood , 104[7]:2163-2171, 2004, PMID: 15213099). We have observed that BCR-ABL-expressing Lin-c-Kit+ Sca-1+ bone marrow (BM) cells function as leukemic stem cells (LSCs). We tested whether LSCs play a role in the acceleration of CML development. We found that the percentage of LSCs in BM of P-selectin−/− CML mice was significantly higher than those in wild type C57BL/6 (B6) CML mice; other BM populations such as CMP, GMP, and MEP had no significant differences between the two groups of CML mice, suggesting that LSCs are responsible for the acceleration of CML disease when P-selectin is deficient. We also analyzed BM of B6 and P-selectin−/− mice and the BM of B6 and P-selectin−/− mice transplanted with BM transfected with empty vector virus. We found that there was no significant difference in the percentage of the stem cell population between the two groups of mice. This suggests that the higher percentage of LSCs in P-selectin−/− CML mice is caused by BCR-ABL but not by transplantation procedure associated with the P-selectin deficiency. Taken together, our results demonstrate that in CML mice, increased LSC population in P-selectin-deficient CML mice is associated with the acceleration of CML development.