Resistant Leukemia Stem Cells Research Articles

Mir-126 Governs Human Leukemia Stem Cell Quiescence and Chemotherapy Resistance

miRNA expression is deregulated in human acute myeloid leukemia (AML), however the impact of altered post-transcriptional programs on the genesis and maintenance of leukemia stem cells (LSC) remains undefined. In order to elucidate the functional role of miRNA in LSC and identify relevant miRNA candidates, we performed global miRNA profiling on sorted cell subpopulations from 16 AML patient and 3 umbilical cord blood samples (Eppert et al, Nature Medicine 2011). Supervised analysis guided by the ability of each sub-population to initiate leukemic engraftment after xenotransplantation into immune-deficient mice generated a unique miRNA signature. miR-126, a miRNA that we previously demonstrated to have a conserved role in maintaining hematopoietic stem cell (HSC) quiescence (Lechman et al. Cell Stem Cell, 2012), was more highly expressed in LSC-enriched fractions and chosen for further validation. To confirm that miR-126 is a bona fide LSC determinant, we utilized a bidirectional lentiviral reporter vector specific for miR-126 (Gentner et al. Science Translational Medicine, 2010) to sort cells from AML patient samples based on miR-126 bioactivity, and demonstrated that all in vivo leukemia-initiating capacity was confined to cells with elevated miR-126 bioactivity. Lentiviral enforced expression of miR-126 in primary AML patient samples significantly increased LSC frequency (3.5-52.3 fold) as assessed by limiting dilution transplantation assays, while diminishing cell cycle entry, differentiation marker expression (CD14,CD15) and colony forming potential. Sponge-mediated knockdown of miR-126 expression resulted in the opposite effects. These findings suggest that high levels of miR-126 bioactivity support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Moreover, by preserving LSC quiescence miR-126 promoted chemotherapy resistance, in part through suppression of CDK3, a gatekeeper of G0 to G1 cell cycle transit. Enforced expression of CDK3 partially rescued the functional consequences of supra-physiological levels of miR-126 bioactivity, rendering previously resistant LSC susceptible to killing by AraC/Daunorubicin combination chemotherapy. Our human LSC miRNA signature, optimized by regression analysis on a cytogenetically normal AML patient cohort, was prognostic for survival in a large independent AML patient cohort (Ley et. al N Engl. J Med, 2013) further validating the clinical significance of miRNA as stem cell determinants. Furthermore, miRNA-126 alone was prognostic for survival in two independent cohorts of AML patients with normal cytogenetics. These data demonstrate a mechanistic role for miR-126 in governing intrinsic LSC properties and establish miR-126 as a critical biomarker for clinical outcome. Disclosures:Wang:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.

The Role Of CD44 Isoform Expression In Niche Resident Chronic Myeloid Leukemia Stem Cell Evolution

IntroductionChronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the BCR-ABL fusion oncogene whose protein product has greatly increased ABL1 kinase activity. Although specific BCR-ABL tyrosine kinase inhibitors (TKIs) and the second generation TKIs like Nilotinib and the dual specific SCR and ABL inhibitor Dasatinib/Sprycel have dramatically improved CML therapy and significantly slowed disease progression by eradicating the bulk of CML cells in the circulation, they frequently fail to eliminate quiescent leukemic stem cells residing in the protective bone marrow niche. Leukemia stem cells (LSC) are able to drive disease relapse and may eventually contribute to the emergence of TKI resistant blast crisis (BC) CML, which is the final phase in the evolution of CML with rapid progression and short survival. CD44 is an adhesion molecule that promotes retention in the niche through adhesion to extracellular matrix components, such as hyaluronic acid and osteopontin. It plays an important role in wound healing and cell migration as well as in tumor invasion and metastasis. Through alternative mRNA splicing several CD44 isoforms exist, some of which are frequently overexpressed by cancer stem cells, including LSCs. The CD44 variant expression pattern on human blast crisis CML LSC, however, had not been elucidated. In this study we aimed to investigate the CD44 transcript variant expression of human blast crisis CML LSC. Methods and ResultsWe performed whole transcriptome RNA sequencing of FACS sorted CML LSCs (Lin-CD34+CD38+) from chronic phase (CP)(n=8) and blast crisis (BC)(n=8), CML patients as well as the normal counterpart from cord blood (CB) (n=3) and adult peripheral blood (NPB)(n=3). A number of CD44 transcript variants were detected: v3, v4 (CD44s), v5, v6, v7, v8 plus additional variants. Earlier data suggest a total of 16 different isoforms of CD44. We found a higher overall variant gene expression of CD44 in BC compared to CP. A higher expression of CD44 transcript variant 3 and CD44 transcript variant 5 was detected in both CP and BC compared to CB and NPB. Specific CD44 transcript variant expression patterns distinguished BC progenitors from CP samples. Using splice isoform specific PCR, we were able to confirm isoform variants that were upregulated in the BC CML samples. We also compared the expression of the CD44 transcript variants in young versus old bone marrow in order to exclude that LSC-specific isoform variant expression was expressed in aged patients. We could not detect such a correlation. ConclusionsThese observations suggest that unique CD44 isoform expression patterns predict progression from CP to BC as well as the generation of TKI resistant LSCs and may be used as biomarkers of response to LSC targeted therapy. Disclosures:Jamieson:J&J, Roche: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees.

Drug resistance of leukemic stem cells mediated by hedgehog signaling pathway

Drug resistance and relapse are the major challenge for current treatment of acute leukemia. It is critical for ultimately curing leukemia to overcome chemoresistance of leukemic stem cells (LSC) and to eradicate LSC. Recent studies have found that abnormal activated Hedgehog (HH) signaling pathway plays an important role in a wide variety of tumors and regulates multi-drug resistance of LSC. This review briefly summarizes the molecular mechanism of HH signal pathway inducing drug resistance of LSC and leading to novel strategies for eradicating LSC.

Canonical NF-κB Signalling Is a Potential Target in FLT3/ITD AML.

AbstractAbstract 2447Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor is common in acute myeloid leukemia (AML) and is associated with a dismal outcome. Despite initial response, FLT3/ITD AMLs often relapse early, suggesting a residual population of resistant leukemia stem cells (LSC). Clinically, FLT3 inhibitors asmonotherapy have yet to improve outcome significantly and therefore, targeting additional pro-survival pathways may be necessary for this group of AML patients. Mice genetically egineered to express a hemizygous FLT3/ITD mutation develop a progressive, fatal, myeloproliferative neoplasm. Lin−cells isolated from the bone marrow of FLT3/ITD or control mice were subjected to gel shift analysis using a radio-labeled NF-kB binding site. This analysis demonstrated high levels of nuclear activation of NF-kB in the FLT3/ITD-expressing cells, suggesting its activation downstream of mutant FLT3 signaling. MV4–11 is a human AML-derived cell line harboring a homozygous FLT3/ITD mutation. Cells expressing high levels of aldehyde dehydrogenase (ALDH) have been shown to be enriched for LSC in primary AML samples and cell lines. High ALDH expressing MV4–11 cells were isolated using FACS and analyzed for NF-kB activation. Western blot analysis demonstrated preferential phosphorylation of NF-kB p65 by activated IKK on Ser536 in this subpopulation, compared with cells with low ALDH activity. These findings indicate activation of NF-kB in MV4–11 LSCs.We wanted to next test the requirement for NF-kB signaling in transformation by FLT3/ITD mutations. NF-kB p65 null mice die in utero. We therefore established C57BL/6 p65(flox/flox);Mx1-Cre mice. Intra-peritoneal injection of pIpC every other day for 7 doses efficiently deletes the RelA/p65 gene, resulting in expression of <1% of the corresponding RNA or protein. Despite effective excision of p65, the mice survive. Bone marrow cells harvested from control or p65(del/del) mice were transduced with a FLT3/ITD-expressing lentivirus and seeded in methylcellulose without cytokines. Equal transduction rate was verified by measurement of GFP expression by flow cytometry. Reproducibly, p65(del/del) marrow transduced with FLT3/ITD was ineffective in forming cytokine independent colonies, in contrast to wild-type marrow (5 +/− 0.6 vs. 55 +/− 6 colonies per 1E5 cells, P<0.001), and the few p65(del/del) colonies that resulted were smaller than those from p65 expressing wild-type marrow cells. Cells transduced with a lentiviral vector expressing GFP but not FLT3/ITD did not form colonies without cytokines, and p65(del/del) marrow formed normal numbers of colonies of normal size and distribution in the presence of IL-3, IL-6, and SCF.Sorafenib inhibits FLT3 signaling and kills MV4–11 cells with an IC50 of approximately 10 nM. Reproducibly, a sub-toxic dose of sorafenib (5 nM) combined with sub-toxic levels of the IKKb inhibitor IMD-0354 (400 nM) resulted in synergistic cell killing as indicated by the calculated combination index of 0.55.Currently, clinical efforts in FLT3/ITD leukemia concentrate on FLT3 inhibition alone. Our data suggest that canonical NF-kB may be an important pathway in FLT3/ITD AML and that simultaneously targeting FLT3 and NF-kB in this disease may be an effective approach. Disclosures:No relevant conflicts of interest to declare.

Effective Elimination of CML Progenitor and Stem Cells Through Combination of α-CD123 Antibody-Dependent Cell-Mediated Cytotoxicity and Tyrosine Kinase Inhibitor Treatment

AbstractAbstract 32Since the introduction of tyrosine kinase inhibitor (TKI) therapy overall survival and complete molecular response rates in chronic phase chronic myeloid leukemia (CP-CML) patients have significantly improved. However, leukemic stem cells (LSCs) and progenitor cells persist and are thought to be responsible for disease progression, development of TKI resistance and disease recurrence after stopping TKI therapy. Protection by cytokines, such as IL-3 and GM-CSF, provides a potential mechanism of LSC resistance. While in acute myeloid leukemia (AML) monoclonal antibody (mAb) targeting of IL-3 receptor α (CD123), a recognized marker for AML LSCs, has been studied in vitro and in vivo, similar investigations have not been undertaken in CML to date. CSL362 is a genetically-engineered form of the specific blocking mAb 7G3 optimized for Fc receptor binding to achieve maximal antibody-dependent cell-mediated cytotoxicity (ADCC) capacity. Here we investigate the expression of CD123 in CD34+ progenitors and CD34+CD38− LSCs, isolated from CP- and blast crisis (BC) - CML patients, and study the benefits of targeting those cells by CSL362 alone and in combination with TKIs.Flow cytometry analysis established significantly elevated expression of CD123 on CD34+CD38− cells from CP-CML (53.0 ± 5.8 %, n=16, p=0.003) and BC-CML (73.2 ± 6.7 %, n=18, p<0.001) patients compared to normal donors (20.3 ± 4.2 %, n=8), with clear increases in CD123 expression with disease progression in matched samples (n=2). Subsequent assessment of apoptosis, colony forming unit (CFU-GM) and long-term culture-initiating cell (LTC-IC) potential confirmed the ability of CSL362 to block IL-3-mediated rescue of TKI-induced cell death. However, in the presence of other cytokines, likely found in the physiological bone marrow microenvironment, this effect was lost.We also demonstrate by lactate dehyrogenase release and clonogenic assays that CML CD34+ cell numbers are significantly reduced, in a dose-dependent manner, by CSL362-induced ADCC employing NK cells from healthy donors (42.4 ± 8.1 % lysis, n=3, and CFU-GM decreased to 30 ± 10.8 % of controls, n=5, p=0.003). In keeping with this, flow cytometry analysis revealed specific elimination of CP- and BC-CML CD123+ CD34+CD38− cells (from 42.9 % to 18.6 %, n=5, p=0.004, and from 71 % to 35.3 %, n=3, p=0.044, respectively). Importantly, autologous CML patient NK cells, collected after achievement of major molecular response, also mediate CSL362-dependent cytotoxicity similar to allogeneic healthy donor NK cells as indicated by equivalent numbers of remaining CFUs (28 ± 6.7 % vs. 34.9 ± 3.4 %, n=5, Fig. A). We further have evidence to suggest preferential elimination of CML over normal LTC-ICs (30.3 ± 9.9 % vs. 62.6 ± 11.2 % remaining, n=3, p=0.096) in the autologous setting. Of clinical importance, the combination of Nilotinib and CSL362 resulted in a significantly greater reduction in CFUs (additive effect) when compared to either agent alone (Fig. B).Taken together these data suggest that selective ADCC-mediated lysis, likely the major mode of action of CSL362 in vivo, efficiently eliminates CML progenitor and stem cells. Promising results evaluating CSL362/TKI combination treatments, with the expectation to further enhance specificity for leukemic while sparing normal progenitor and stem cells as indicated from preliminary experiments, warrant further studies. [Display omitted] Disclosures:Nievergall:CSL Ltd: Research Funding. White:BMS: Research Funding; CSL Ltd: Research Funding; Novartis Oncology: Honoraria, Research Funding. Ramshaw:CSL Ltd: Research Funding. Busfield:CSL Ltd: Employment. Vairo:CSL Ltd: Employment. Lopez:CSL Ltd: Research Funding. Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Ltd: Research Funding. Hiwase:CSL Ltd: Research Funding.

Sulforaphane Potentiates the Efficacy of Imatinib against Chronic Leukemia Cancer Stem Cells through Enhanced Abrogation of Wnt/β-Catenin Function

Sulforaphane (SFN) has been indicated for the prevention and suppression of tumorigenesis in solid tumors. Herein, we evaluated SFN's effects on imatinib (IM)-resistant leukemia stem cells (LSCs). CD34(+)/CD38(-) and CD34(+)/CD38(+) LSCs were isolated from KU812 cell line flowcytometrically. Isolated LSCs showed high expression of Oct4, CD133, β-catenin, and Sox2 and IM resistance. Differentially, CD34(+)/CD38(-) LSCs demonstrated higher BCR-ABL and β-catenin expression and imatinib (IM) resistance than CD34(+)/CD38(+) counterparts. IM and SFN combined treatment sensitized CD34(+)/CD38(-) LSCs and induced apoptosis, shown by increased caspase 3, PARP, and Bax while decreased Bcl-2 expression. Additionally, the combined treatment reduced BCR-ABL and β-catenin and MDR-1 protein expression. Mechanistically, IM and SFN combined treatment resensitized LSCs by inducing intracellular reactive oxygen species (ROS). Importantly, β-catenin-silenced LSCs exhibited reduced glutathione S-transferase pi 1 (GSTP1) expression and intracellular GSH level, which led to increased sensitivity toward IM and SFN. We demonstrated that IM and SFN combined treatment effectively eliminated CD34(+)/CD38(-) LSCs. Since SFN has been shown well tolerated in both animals and human, this regimen could be considered for clinical trials.

Biological therapy and the immune system in patients with chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells that has been recognized as a disease responsive to immunotherapy. Despite the huge success of the tyrosine kinase inhibitors (TKIs), CML remains for the most part incurable, probably due to treatment resistance of leukemic stem cells, which are responsible for rapid disease relapse after discontinuation of therapy. Only allogeneic stem cell transplantation enables disease eradication. In addition to the Bcr-Abl1 oncoprotein, TKIs also inhibit off-target kinases (e.g. c-kit, Src, Tec), some of them having physiological functions in immune responses. In vitro studies have implied immunomodulatory effects of TKIs and interferon-alpha (IFN-α), but comprehensive information from in vivo analyses is missing. This review summarizes the recent advances in the field of immunology of CML, including basic information about leukemia-associated antigens and peptide vaccines, that could lead to the incorporation of TKIs and IFN-α in future therapeutic, potentially curative, interventions for CML.

New insights into antigen specific immunotherapy for chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a stem cell disease in which BCR/ABL plays an important role as an oncoprotein and a molecular and immunogenic target. Despite the success of targeted therapy using tyrosine kinase inhibitors (TKIs), CML remains largely incurable, most likely due to the treatment resistance of leukemic stem cells. Several immunotherapies have been developed for CML in different stages and relapse after allogeneic stem cell transplantation. In the this review, several specific immunotherapeutic approaches for CML, including vaccination and adoptive cellular immunotherapy, are discussed along with results from clinical trials, and the value of such immunotherapies in the era of imatinib and leukemia-associated antigens (LAAs), which are capable of inducing specific T cell responses and are appropriate target structures for the immunological targeting of CML cells, are also summarized.

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.

Assembling defenses against therapy-resistant leukemic stem cells: Bcl6 joins the ranks

The resistance of leukemic stem cells in response to targeted therapies such as tyrosine kinase inhibitors (TKIs) relies on the cooperative activity of multiple signaling pathways and molecules, including TGFβ, AKT, and FOXO transcription factors (TFs). B cell lymphoma 6 (BCL6) is a transcriptional repressor whose translocation or mutation is associated with diffuse large BCL. New data now show that BCL6 is critical for the maintenance of leukemias driven by the BCR-ABL translocation (Philadelphia chromosome), suggesting that BCL6 is a novel, targetable member of the complex signaling pathways critical for leukemic stem cell survival.

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

Targeting of Leukemia-Initiating Cells to Develop Curative Drug Therapies: Straightforward but Nontrivial Concept

The concept of leukemic stem cells (LSC) is increasingly employed to explain the biology of various myeloid neoplasms and to screen for essential targets, with the hope to improve drug therapy through elimination of disease-initiating cells. Although the stem cell hypothesis may apply to all neoplasms, leukemia-initiating cells have so far only been characterized in some detail in advanced acute (AML) and chronic myeloid leukemia (CML). An intriguing observation is that although expressing various targets, LSC often remain unresponsive against most drugs, presumably because of 'intrinsic' resistance. Moreover, LSC represent heterogeneous populations of cells, grow in separate subclones, and acquire numerous defects, which points to substantial genetic instability and stem cell plasticity. The situation is complicated by the fact that stem cell evolution is a step-wise process with variable latency periods, so that many LSC-derived subclones remain small (undetectable) at diagnosis, but later, during therapy, may expand to a dominant clone and clinically overt relapsing disease. Finally the interaction between LSC and the microenvironment may contribute to stem cell function and LSC resistance. Taking all these considerations into account, the application of broadly acting targeted drugs and of drug combinations has been proposed in order to better suppress or even eliminate LSC in AML and CML. The current article provides a summary of our knowledge on LSC in various myeloid neoplasms with special reference to novel arising treatment concepts.

β-Catenin Suppression Targets Imatinib Resistant Leukemia Stem Cells In Mice with BCR-ABL Induced Myeloproliferative Disease

AbstractAbstract 93CML is driven by the oncogenic fusion kinase BCR-ABL which is capable of corrupting and transforming hematopoietic stem cells (HSC) and thus inititating myeloproliferative disease (MPD). While imatinib (IM) has become an effective treatment for CML, the leukemia stem cells (LSC) which reside in the bone marrow (BM) remain largely unaffected by imatinib treatment. As LSC rely on their ability to self-renew, several genes/pathways responsible for self-renewal properties have been investigated as potential targets of LSCs in myeloid neoplasia. Recent studies clearly indicate that canonical Wnt/β-catenin-signaling plays a pivotal role in initiation and development of BCR-ABL induced MPD (Zhao, Cancer Cell 2009; Hu, Leukemia 2009). However, the impact of β-catenin (Ctnnb1) on the maintenance of LSC in established disease, which more accurately models the clinical scenario, has not been extensively studied.To address the impact of targeting Ctnnb1 in CML-LSC, we used a conditional Ctnnb1 knockout mouse with an estrogen-receptor driven Cre-recombinase as BM-donors. CML was induced by retroviral transduction of either Ctnnb1flox/flox ER-Cre+, Ctnnb1flox/wt ER-Cre+, or Ctnnb1wt/wt ER-Cre+ murine bone marrow with a virus encoding BCR-ABL (MSCV-BCR-ABLp210-GFP), and injection of transduced cells into wildtype syngeneic recipient mice. Administration of tamoxifen to recipient mice with CML led to excision of Ctnnb1 in the Ctnnb1flox/flox homozygous or Ctnnb1flox/wt heterozygous cells.Interestingly genetic inactivation of Ctnnb1 after onset of CML in primary recipient mice resulted only in minor prolongation of survival. The animals died due to organ infiltration (primarily lung) by differentiated progenitor cells that were presumably not affected by Ctnnb1 deletion. However, in the BM and peripheral blood, a clear reduction of GFP+ cells was evident in heterozygous - and even more pronounced - in homozygous animals, indicating a potential effect on more immature cells and perhaps LSC. To determine if LSC were compromised and to investigate the efficacy of a combination treatment with IM, we used a serial BM-transplantation assay. Consistent with previously published data, we found that leukemia cells in the Sca-1/GFP+ fraction are able to re-establish CML in secondary recipient mice and thus contain LSC. IM treatment alone led to a relative increase of Sca-1/GFP+ LSC over time. However, we found a significant reduction of Sca-1/GFP+ bone marrow cells after inactivation of Ctnnb1 during IM-treatment. Mice transplanted with Ctnnb1flox/flox ER-Cre+ CML cells had on average 0.025% Sca-1/GFP+ (total of 7,375 cells/mouse) cells in the bone marrow after treatment of the recipient mice with IM and tamoxifen, mice that received Ctnnb1flox/wt ER-Cre+ CML had 0.036% Sca-1/GFP+ cells (total of 10,584 cells/mouse) and mice that received Ctnnb1wt/wt ER-Cre+ CML had 0.075% Sca-1/GFP+ cells (total of 24,375 cells/mouse). As indomethacin has recently been shown to inhibit Ctnnb1 in hematopoietic and AML stem cells (Goessling, Cell 2009; Wang, Science 2010), we aimed to investigate the efficacy of indomethacin combined with IM. We were able to demonstrate a decrease in Ctnnb1 protein after indomethacin treatment of human cell lines and transformed murine BM cells by either immunoblot, flow-cytometry or immunofluorescence. Moreover, we were able to show synergy between indomethacin and IM co-treatment which produced almost complete abrogation of Ctnnb1 protein. IM/Indomethacin co-treatment of mice with CML decreased the Kit+Sca1+ fraction of the Lin-GFP+ bone marrow population to 0.92% (1799 cells/mouse) whereas treatment with IM alone was similar to untreated mice with 2.8% of the Lin-GFP+ population being Kit+Sca1+ (22,601 cells/mouse). Importantly, transplantation of the bone marrow from treated mice into tertiary recipients showed significantly prolonged survival of tertiary recipient mice that received bone marrow from donors treated with the combination therapy (IM/INDO) compared to IM/DMSO controls (p=0.0038*).In summary, we demonstrate that inhibiting Ctnnb1 by genetic inactivation or drug treatment is an effective combination therapy with imatinib mesylate and targets CML leukemia stem cells. This provides strong evidence for the development and clinical use of β-catenin-targeted therapies in CML. Disclosures:No relevant conflicts of interest to declare.

Evaluation of Residual CD34+/Ph+ Stem Cells In Chronic Myeloid Leukemia Patients In Complete Cytogenetic Response during First Line Nilotinib Therapy.

AbstractAbstract 3413 IntroductionImatinib mesylate is highly effective in inducing rapid hematologic and cytogenetic responses in the vast majority of chronic myeloid leukemia (CML) patients. Yet, discontinuation of treatment is associated with disease relapse probably due to the persistence of resistant leukemic stem cells representing a reservoir of the disease. On this regard it has been reported that BCR-ABL positive progenitor cells can still be detected in patients in complete cytogenetic response (CCyR) after short term of imatinib treatment (Bhatia R, et al. Blood. 2003;101:4701-4707) but also after a stable long lasting CCyR (Bocchia M, et al. Leukemia. 2008;22:426-428). Compared to imatinib, the second generation Tyrosin Kinase Inhibitor (TKI) nilotinib appears to eradicate more rapidly the bulk of CML cells, inducing high rate of CCyR and major molecular response (MMolR) after a very short period of treatment (78% CCyR and 52% MMolR at 3 months) (Rosti G, et al. Blood. 2009;114(24):4933-8). Despite nilotinib is a more potent TKI, it didn't appear to be more effective in eliminating in vitro CML progenitors than imatinib (Konig H, et al Leukemia. 2008;22:748-755). Up to date no data evaluating the persistence of Ph+ stem cells in early chronic phase CML patients during first line treatment with nilotinib have been reported. Patients, materials and methodsWe investigated the presence of residual CD34+/Ph+ cells in 24 CML patients in CCyR during first line nilotinib treatment. Patients were enrolled in 2 clinical trials (GIMEMA CML0307 and CAMN107A2303) and evaluation of residual leukemic stem cells was performed during a routine bone marrow aspirate after receiving specific patients informed consent. Bone marrow purified CD34+ cells were evaluated for BCR-ABL fusion gene by fluorescent in situ hybridization (FISH) analysis. A minimum of 100 interphase nuclei of purified CD34+ cells was considered optimal for FISH analysis. ResultsAll 24 patients have been treated exclusively with nilotinib since diagnosis (17/24 at 400mg bid; 5/24 at 300mg bid; 2/24 at 400mg/day). At the time of analysis all 24 patients were in CCyR for a median time of 27 months (range 6–29) after being treated for a median time of 30 months (range 9–30) with this second generation TKI. Regarding molecular response 20/24 (83%) were in MMolR while only 1/24 (4%) was in CMolR. Harvest, purification and subsequent FISH analysis of bone marrow CD34+ cells was optimal in 15/24 (63%) patients, suboptimal in 5/24 (21%) patients (less than 100 interphase nuclei analyzed) and not adequate in 4/24 (16%) patients (less than 50 interphase nuclei). With respect to leukemic stem cells, residual CD34+/Ph+ cells were found only in 1/20 (5%) evaluable patients. Of note, in this patient 140 CD34+ interphase nuclei were analyzed and only 1 was found bcr-abl positive (0.7%). ConclusionOur study shows for the first time that in patients in CCyR during front line Nilotinib treatment residual CD34+/Ph+ stem cells are very rarely detected. These results quite differ from what was previously found in imatinib treated patients (Bocchia M, et al. Leukemia. 2008;22:426-428). In fact in the present series, only 1/20 (5%) patients treated with nilotinib in CCyR for a median time of 27 months showed residual CD34+/Ph+ cells, while in our prior study residual leukemic CD34+ cells were still detectable in 14/31 (45%) imatinib treated patients in stable CCyR (median of 39 months). Despite the limited number of patients studied, this novel evidence may support the better short term clinical results observed with nilotinib as first line treatment in CML. Disclosures:Rosti:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau.

Resistance to Imatinib: Mutations and Beyond

Mechanisms of resistance to the tyrosine kinase inhibitor (TKI) imatinib had been modeled in vitro even prior to the first reports of clinical resistance in patients with chronic myeloid leukemia (CML). The discovery that BCR-ABL is reactivated at the time of resistance and the unveiling of point mutations within the kinase domain of BCR-ABL as a major resistance mechanism have driven the development of second-generation TKIs. These agents are effective in a significant proportion of patients who fail to respond to imatinib. Clinical practice guidelines recommend using the BCR-ABL mutation genotype to aid selection of second-line treatment. Although kinase domain mutations are undoubtedly relevant to drug resistance, recent data suggest that additional resistance mechanisms must be operational in patients with and without kinase domain mutations. Clonal chromosomal evolution, BCR-ABL amplification, pharmacogenomic variations, or activation of signaling shortcuts have all been implicated in drug resistance, but their precise contributions to resistance remain to be determined. Additionally, lack of adherence to prescribed medication is likely to set the stage for resistance development. An area of intense research is primary resistance of leukemic stem cells (LSCs), which are thought to cause minimal residual disease to persist despite sustained treatment. The intent of this review is to shed light on the various aspects of TKI resistance in CML with respect to their biology and clinical implications.

Abstract A29: High aldehyde dehydrogenase activity is general marker for normal hematopoietic stem cells but not leukemic stem cells in acute myeloid leukemia

Abstract Only a minority of cells, the leukemic stem cells (LSCs), within Acute Myeloid Leukemia (AML) are typically responsible for tumor growth and maintenance. Many patients experience a relapse after therapy which originates mainly from the outgrowth of therapy resistant LSC. Therefore, eradication of the LSCs is likely necessary to cure leukemia. Both the normal hematopoietic stem cells (HSCs) and LSCs co-exist in the bone marrow (BM) of leukaemia patients and therefore success of an anti-stem-cell strategy relies on specific induction of LSC death while sparing the normal HSC. In AML, apart from the CD34+CD38- and the side population (SP) compartment, the high aldehyde dehydrogenase (ALDH) activity compartment contains the LSCs. ALDH is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes. Recent data has shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blast cells. High ALDH activity results in resistance to alkylating agents such as the active derivatives of cyclophosphamide. We have previously shown that CD34+CD38- and SP LSCs can be identified and discriminated from HSCs using stem cell-associated cell surface markers, including C-type lectin-like molecules (CLL-1), lineage markers, such as CD7, CD19, and CD56 and recently cell size characteristics as measured by flow cytometry. Here we have analyzed ALDH activity in normal HSCs and LSCs, both present within the same BM, in 23 AML patient samples. In 7 AML cases, a high SSCloALDHbr cell population was identified (&amp;gt;5%). We show that in 9 BM AML samples, defined as CD34min (ie &amp;lt;1% CD34+ cells, in which the CD34+CD38-cells are all normal HSC), the ALDH activity is lower in the LSC then in the HSC. The activity of the normal HSC within this AML BM is identical as the ALDH activity of normal HSC in NBM of healthy donors. In 9 BM AML patients, defined as CD34+ and with both normal and leukemic CD34+CD38-present, we show that the marker positive CD34+CD38-LSCs have lower ALDH activity then the marker negative CD34+CD38-HSCs. Altogether, we show that, although malignant AML blast cells have varying ALDH activity, a common feature of all AML cases is that the normal HSCs that co-exist with leukemic (stem) cells in the BM of AML patients have a higher ALDH activity as compared to their malignant counterparts. In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM at diagnosis. Consequently, AML patients with high ALDH activity in the normal HSC might benefit from treatment with alkylating agents such as mafosfamide, whereby the difference between the ALDH activity in LSC and HSC defines the therapeutic window. At present, drugs, known to be dependent on low ALDH for proper activity, are tested for their LSC specific killing while sparing the normal HSC. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A29

Differential destruction of stem cells: implications for targeted cancer stem cell therapy.

Cancer stem cells represent a novel therapeutic target. The major challenge in targeting leukemic stem cells (LSC) is finding therapies that largely spare normal hematopoietic stem cells (HSC) while eradicating quiescent LSCs. We present a mathematical model to predict how selective a therapy must be to ensure that enough HSCs survive when LSCs have been eradicated. Stem cell population size is modeled as a birth-death process. This permits comparison of LSC and HSC eradication times under therapy and calculation of the number of HSCs at the time of LSC eradication for varied initial population sizes and stem cell death rates. We further investigate the effects of LSC quiescence and resistance mutations on our predictions. From a clinical point of view, our models suggest criteria by which cancer stem cell therapy safety can be assessed. We anticipate that in conjunction with experimental observation of cancer stem cell killing rates, our results will be useful in screening targeted therapies for both hematologic and solid tumor malignancies.

High Aldehyde Dehydrogenase Activity (ALDH) Is a General Marker for Normal Hematopoietic Stem Cells but Not Leukemic Stem Cells in Acute Myeloid Leukemia (AML). .

Abstract 4035Poster Board III-971Only a minority of cells, the leukemic stem cells (LSCs), within AML are responsible for tumor growth and maintenance. Many patients experience a relapse after therapy which is thought to originate from the outgrowth of therapy resistant LSC. Therefore, eradication of the LSCs is likely necessary to cure AML. Both the normal hematopoietic stem cells (HSCs) and LSCs co-exist in the bone marrow (BM) of leukemia patients and therefore success of an anti-stem-cell strategy relies on specific induction of LSC death while sparing the normal HSC. In AML, apart from the CD34+CD38- and the side population (SP) compartment, the high ALDH activity compartment contains the LSCs. The SP and ALDH defined compartments may include both CD34+ and CD34- HSCs and LSCs. ALDH is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes and high ALDH activity results in resistance to alkylating agents such as the active derivatives of cyclophosphamide. Recent data has shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blast cells. In view of the applicability of LSC specific therapies the detoxification by ALDH might be of importance. Therefore, a difference in ALDH activity between the normal HSC and the malignant LSC might be used to preferentially kill the LSC and spare the HSC.We have previously shown that CD34+CD38- and SP LSCs can be identified and discriminated from HSCs using stem cell-associated cell surface markers, including C-type lectin-like molecules (CLL-1), lineage markers, such as CD7, CD19, and CD56 and recently cell size characteristics as measured by flow cytometry (Terwijn, Blood 111: 487,2008). Here we have analyzed ALDH activity in 23 AML cases. In 7 AML cases, a high SSCloALDHbr cell population was identified (median: 10,9%, range 5,24-15,29%). In 16 cases there were rare (<5%) SSCloALDHbr cells.We have analyzed ALDH activity in aberrant marker defined HSCs and LSCs, both present within the same BM samples in 18 AML patients (summarized in Figure 1). In 9 BM AML samples, defined as CD34-, the CD34+ compartment contained only normal CD34+CD38- HSCs. The ALDH activity in the CD34- cells, which includes by definition in this AML subgroup the LSC, is a factor 4,4 (range 1,7-18,9) lower than in the HSC (Figure 1, panel 1). The ALDHbrSSClo cells present in these CD34- AML cases contained both normal CD34+ and CD34- cells. The activity of the normal HSC within this AML BM is similar to that of the normal HSC in NBM of healthy donors (Figure 1, panel 3).In addition, 9 BM AML patients, defined as CD34+ AML and with both marker negative, normal HSCs and marker positive LSCs present, were analyzed for ALDH activity. We show that the marker positive CD34+CD38- LSCs have 7,7 fold (range 1,73-29,2 fold) lower ALDH activity than the marker negative CD34+CD38- HSCs (Figure 1, panel 2). Altogether, we show that, although malignant AML blast cells have varying ALDH activity, a common feature of all AML cases is that the normal HSCs that co-exist with leukemic (stem) cells in the BM of AML patients have a higher ALDH activity as compared to their malignant counterparts (summarized in figure 1).In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM (irrespective of AML phenotype, CD34+ or CD34-) at diagnosis. Consequently, AML patients with high ALDH activity in the normal HSC might benefit from treatment with alkylating agents such as cyclophosphamide, whereby the difference between the ALDH activity in LSC and HSC defines the therapeutic window. At present, drugs, known to be dependent on low ALDH for proper activity, are tested for their LSC specific killing while sparing the normal HSC. Additionally, transcriptional profiles are obtained from purified ALDH+ HSC and ALDH- LSC. This will enable us to use this general discriminating property to identify molecules that differ between the LSC and HSC and can function as LSC specific therapeutic targets. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High Expression of MDR1 and BCRP1 in Leukemic Stem Cells Correlates with Reversible Drug Resistance Ex Vivo and Predicts Failure of Remission Induction Chemotherapy in Patients with Acute Myeloid Leukemia.

Although overexpression of members of the ATP-Binding-Cassette (ABC) transporter superfamily has been implicated in chemotherapy failure in acute myeloid leukemia (AML) in some studies, results have been conflicting. Previously we found no consistent difference in pre-treatment ABC transporter expression when comparing total blasts of 18 patients achieving complete remission (CR) and 13 patients who were refractory to induction chemotherapy (NR). However, among the CD34+CD38− AML cells (enriched for candidate “leukemic stem cells” which engraft in immunodeficient mice), as assessed by quantitative RT-PCR, elevated expression of MDR1 and/or BCRP1, two ABC transporters associated with drug resistance, was found in 8/10 NR patients as compared to 0/7 CR patients. No difference between CR and NR samples was observed in the more differentiated CD34+CD38+ or CD34− AML cells while none of the subpopulations showed a significant difference in MRP1 expression between CR and NR patient samples. To determine if this difference in gene expression was associated with altered functional activity of MDR1/BCRP1, the relative sensitivity of subpopulations of AML cells to induction of apoptosis following ex vivo treatment with daunorubicin with or without ABC inhibitors (PSC-833 and Verapamil) was assessed. All subpopulations of AML cells isolated from 2 CR patients showed similar drug sensitivity profiles (IC50 <0.04 μg/ml). In contrast, CD34+CD38− cells from 6 NR patients were more resistant to daunorubicin-induced apoptosis (p<0.05 for comparison of IC50s between CR and NR samples). ABC transporter inhibition in CD34+CD38− cells did not change daunorubicin sensitivity in CR samples, but increased drug sensitivity in a dose-dependent manner in NR samples, particularly in the CD34+CD38− fraction. This suggests a role for ABC transporters in sustaining a population of chemotherapy resistant leukemic stem cells which may allow survival of the malignant clone in patients following induction therapy. If the value of measuring MDR1/BCRP1 expression among these cells in predicting chemotherapy outcome is confirmed in a larger patient population, it may be possible to use such analysis to modify therapy for individual AML patients to overcome drug resistance.

Breast Cancer Resistance Protein in Drug Resistance of Primitive CD34+38− Cells in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete chemotherapeutic eradication of leukemic CD34+38- stem cells is likely to result in disease relapse. The purpose of this study was to investigate the role of the breast cancer resistance protein (BCRP/ATP-binding cassette, subfamily G, member 2) in drug resistance of leukemic stem cells and the effect of its modulation on stem cell eradication in AML. BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody) and the effect of its modulation (using the novel fumitremorgin C analogue KO143) on intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in leukemic CD34+38- cells. BCRP was preferentially expressed in leukemic CD34+38- cells and blockage of BCRP-mediated drug extrusion by the novel fumitremorgin C analogue KO143 resulted in increased intracellular mitoxantrone accumulation in these cells in the majority of patients. This increase, however, was much lower than in the mitoxantrone-resistant breast cancer cell line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage due to the presence of additional drug transport mechanisms, among which ABCB1 and multiple drug resistance protein. In line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38- cells. These results show that drug extrusion from leukemic stem cells is mediated by the promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather than modulation of single mechanisms, is therefore likely to be required to circumvent drug resistance and eradicate leukemic stem cells in AML.