ALBERT

Description: Recently, it has been demonstrated that the proapoptotic protein BIM showed a deletion polymorphism at exon 3 in eastern Asian population, and some CML patients with the BIM deletion polymorphism are resistant to imatinib treatment (Ng et al. Nature Medicine, 2012). More recently, a BIM single nucleotide polymorphism (SNP) at exon 8 (c465C〉T) has also been found in French CML patients and this SNP is associated with not only imatinib resistance but also the presence of BCR-ABL mutations (Mahon et al. ASH abstract, 2012). We aimed to investigate a possible association between such genetic variations of BIM and clinical manifestation in Japanese CML patients who experienced undetectable minimal residual disease (UMRD: so-called CMR4.5). In this study, we newly analyzed BIM SNP (c465C〉T) in 47 CML-UMRD patients with known BIM deletion polymorphism status (Katagiri et al. Br J Haematol, 2013). Twenty normal subjects were used as controls. The frequency of either BIM SNP at exon 8 or BIM deletion polymorphism did not deviate from the normal subjects in the Japanese population (P = 0.7597 and P = 0.2880, respectively). None of the subjects showed both BIM SNP at exon 8 and BIM deletion polymorphism concomitantly. We then compared the clinical features among 3 CML-UMRD groups: patients with BIM SNP, patients with BIM deletion polymorphism, and patients who showed neither BIM SNP nor BIM deletion polymorphism (no genetic variations). The frequency of CML patients who maintained 400 mg imatinib dose until stopping was significantly higher in those without genetic variations than in those with BIM SNP or BIM deletion polymorphism (P = 0.0002). Moreover, the frequency of CML patients who switched to second tyrosine kinase inhibitors (2nd TKIs) was significantly higher in those with BIM SNP or BIM deletion polymorphism than in those without such polymorphisms (P = 0.0055).Number of CML patientsMaintained IM 400 mgChange of imatinib dose2nd TKIs switchingBIM SNP (c465C〉T)11/474/115/112/11BIM deletion polymorphism6/471/63/62/6BIM SNP or deletion polymorphism17/475/178/174/17No BIM genetic variationss30/4725/305/300/30 This is apparently the first study to circumstantiate the BIM genetic variants in Japanese CML patients with UMPD. Although the number of patients is small, our results suggest that CML patients without BIM deletion polymorphism/SNP could be maintained under standard imatinib dose without switching to 2nd TKIs, and thereby, have a possibility to stop TKIs therapy. Disclosures: Ohyashiki: Norvartis KK: Research Funding, Speakers Bureau; Bristol Meyer Squibe KK: Research Funding, Speakers Bureau.

Description: Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy (e.g. imatinib, nilotinib and dasatinib) has improved the survival of Ph-positive leukemia patients. However, despite the impressive efficacy of these agents, disease relapse has been observed in clinically. Mutations in the BCR-ABL kinase domain can cause of ABL TKI resistance. In particular, one of the BCR-ABL kinase domain mutations (e.g. T315I) is associated with a high level of resistance to all available ABL TKIs. Ponatinib (formally, AP24534) is a multi-target TKI. Recently, in the PACE (Ponatinib Ph+ acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML) Evaluation) trial, ponatinib showed significant efficacy against Ph-positive leukemia in patients with multi-resistant T315I mutations. However, in some patients, especially those with Ph-positive ALL, ponatinib resistant clones were identified. Omacetaxine mepesuccinate, formally known as homoharringtonine is a natural alkaloid obtained from various Cephalotaxus species. Omacetaxine is a first-in-class cephalotaxine in clinical development as anti-leukemic therapy. Omacetaxine acts by binding to the A-site cleft of ribosomes and thereby transiently inhibits protein synthesis. Omacetaxine was approved for the treatment adult patients with chronic or the accelerated phase of CML resistant to other therapies. We investigated the efficacy of omacetaxine against ponatinib resistant Ph-positive cells. Ba/F3 ponatinib resistant cells (Ba/F3 ponatinib-R) have three BCR-ABL point mutations (Y253H, E255K and T315I: data not shown). With 72 h omacetaxine treatment, the cell growth of Ba/F3 ponatinib-R and Ph-positive ALL cell line was significantly reduced even at a low concentration and it is also effective to the other hematological malignancies such as acute myeloid leukemia. In contrast, Ba/F3 ponatinib-R was resistant to ponatinib. With 48 h treatment, omacetaxine dependent apoptosis was increased. Although anti-apoptotic proteins were not increased in this cell line compared to parental cells, as compound mutations such as E255V/T315I confer high-level resistance to ponatinib, these three point mutant was associated with vitro resistance to ponatinib. We also examined intracellular signaling. The phosphorylations of BCR-ABL and a down-stream molecule, Crk-L, were decreased. Protein expressions of BCR-ABL and Crk-L were also decreased. However, caspase-3 and cleaved Poly (ADP-ribose) polymerase (PARP) levels were significantly increased in low concentration. In a previous study, omacetaxine was shown to induce apoptosis in leukemic cells due to a selective decrease in short-lived proteins. We found that omacetaxine reduced the expression of BCR-ABL and heat shock protein 90(HSP90) which is stabilize BCR-ABL protein. We also found that omacetaxine reduced the expression of anti-apoptotic protein, Bcl-2. The protein expression of c-myc was also reduced. We next examined a ponatinib resistant primary Ph+ ALL and chronic phase CML samples. The ponatinib resistant primary cells have several BCR-ABL point mutations (e.g. Q252H, E255K/V, and T315I). We found the growth of primary cells was resistant to ponatinib but to be reduced after omacetaxine treatment and similar signaling events were occurred in OM-treated primary ALL cells. Omacetaxine is an inhibitor of protein synthesis. Because omacetaxine inhibits the BCR-ABL, Bcl-2 and HSP90 pathways in BCR-ABL positive leukemia cells through reduced the levels of these proteins, Omacetaxine has anti-tumor activity and promotes apoptosis. Our findings suggest that omacetaxine may benefit patients with leukemic BCR-ABL mutant cells, possibly allowing ponatinib resistant clones to be overcome. Disclosures: Ohyashiki: Novartis: Honoraria, Research Funding.

Description: Hematopoietic stem cells and leukemic stem cells share common features, including self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. Despite these similarities, several stemness factors, such as Hedgehog, Wnt, Notch, and Dopamine Receptor show differential activation in normal versus leukemia stem cells. Using a high-throughput discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells, the norvel dopamine receptor antagonist, thioridazine, was identified from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal (Cell 149, 1284, 2012). In the present study, we investigated the molecular mechanisms by which dompamine receptor antagonist, thioridizine, regulates the self-renewal of primary BCR-ABL1 positive leukemia cells in vivo. To identify the leukemia-propagating cell fraction of BCR-ABL1-positive leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n=1; T315I BCR-ABL1) or ponatinib-resistant Ph-positive acute lymphoblastic leukemia (n=2, Y253H/E255K/T315I BCR-ABL1 and T315I BCR-ABL1) into NOD/SCID/IL-2γc-/- mice. The cell fractions with CD34+CD38- CD19+and CD34+CD38+CD19+ could self-renew and transfer the leukemia in NOD/SCID mice. To investigate the effects of the domamine receptor antagonist on self-renewal and the relevance as a therapeutic target in ABL-tyrosine kinase-resistant BCR-ABL1 positive leukemia, we examined the activity of thioridazine against CD34+CD38-CD19+, CD34+CD38+CD19+ fractions transferred NOD/SCID mice in vivo. NOD/SCID mice were injected intravenously with BCR-ABL1 positive cells then treated with thioridazine (20 mg/kg; p.o.) for 28 days. All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with thioridazine reduced the population of CD34+CD38- positive cells. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of leukemia cells into secondary recipients. Following 30 days, all mice received BCR-ABL1 cells from vehicle treated mice engrafted with leukemia. In contrast, leukemia engraftment was not detected in recipient mice (n=6) from thioridazine-treated donors. These results demonstrate the persistent effects of domapine receptor signaling inhibition on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of dompamine receptor pathway modulation on in vitro clonogenic growth. CD34+CD38-CD19+ cells from T315I BCR-ABL1 (n=2) and WT-BCR-ABL1 (n=1) cells were treated with 5 μM of thiorizaxine for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. Dopamine receptor pathway inhibition by thioridazine had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by thioridazine (p

Description: Abstract 1333 Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor, imatinib has demonstrated the potency against CML patients. However, resistance to imatinib can develop in CML patients due to BCR-ABL point mutations. One of T315I mutation is resistant to currently available ABL tyrosine kinase inhibitors. Therefore, new approach against T315I mutant may improve the outcome of Ph-positive leukemia patients. Aurora kinases are serine/threonine kinases and upregulated in many malignancies including leukemia, and play an important role in cell cycle control and tumor proliferations. Because Aurora kinases are overexpressed in leukemia cells, Aurora kinases may present attractive targets for leukemia treatment. One of Aurora kinase inhibitor, MLN8237 (alisertib) is an oral and selective Aurora kinase A inhibitor and is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies. We suggested that alisertib mediated inhibition Aurora kinase activity and in combination with ponatinib, also known as AP24534 may abrogate the proliferation and survival of Ph-positive cells including T315I mutation. In this study, we investigated the combination therapy with a ponatinib and an alisertib by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, ponatinib resistant Ba/F3 cells and T315I primary sample. Protein expression of Aurora A and B were increased in Ph-positive leukemia cells. 72 hours treatment of alisertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. Alisertib also induced cell cycle arrest. The treatment of ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of alisertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We investigated the alisertib activity against T315I positive cells. Alisertib potently induced cell growth inhibition of Ba/F3 cells ectopically expressing T315I mutation and induced cell cycle arrest. We investigated the efficacy between ponatinib and alisertib by using these cell lines. Combined treatment of Ba/F3 T315I cells with ponatinib and alisertib caused significantly more cytotoxicity than each drug alone. Ponatinib and alisertib were also effective against T315I primary samples. We examined the intracellular signaling of alisertib. Phosphorylation of Aurora A was inhibited in a time dependent manner. We also found the phosphorylation of histone H3 was also reduced in a dose dependent manner suggested that high concentration of alisertib also inhibits Aurora B activity. We next investigated by using ponatinib resistant Ba/F3 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of alisertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells in the dose dependent manner. Alisertib induced cell cycle arrest in ponatinib resistant cells. Combined treatment of Ba/F3 ponatinib resistant cells with ponatinib and alisertib caused significantly more cytotoxicity. To assess the activity of alisertib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with 1×107 Ba/F3 T315I cells. A dose of 30 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of alisertib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed indicating the feasibility of alisertib combination strategies in the clinic. Data from this study suggested that administration of the ponatinib and Aurora inhibitor, alisertib may be a powerful strategy against BCR-ABL mutant cells including T315I. Disclosures: No relevant conflicts of interest to declare.

Description: Background: The inhibition of BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has markedly improved the prognosis of chronic myeloid leukemia (CML). Recently, it has been recognized that some CML patients with a complete molecular response (CMR) are able to maintain treatment-free remission (TFR) after discontinuation of TKIs. However, no predictive prognostic factors for successful discontinuation of the treatment have yet been identified. We set out to further clarify the role of predictive biomarkers in molecular relapse and non-relapse after ABL TKI discontinuation. Materials and methods: Patients in sustained CMR (MR 4.5) undergoing TKI therapy were eligible for inclusion in the study. Molecular relapse was defined as loss of major molecular response (MMR) of at least one point. Genomic DNA was obtained from whole blood using a DNA Extractor WB Kit (Wako, Osaka, Japan), and was subjected to polymerase chain reaction (PCR) amplification using primers designed to detect a deletion site (2903 bp) in intron two of the BCL2L11 gene (forward: 5′-AATACCACAGAGGCCCACAG-3′; reverse: 5′-GCCTGAAGGTGCTGAGAAAG-3′) and JumpStart RedAccuTaq LA DNA polymerase (Sigma Aldrich, St. Louis, MO, USA). Results: 32 CML patients (17 men, 15 women, median age 58.4 years) were included in this study (Sokal category; low 24, intermediate 7, high 1). Six patients were treated with IFNα before TKI treatment, and 3 were treated with IFNα after stopping TKI. Median duration from TKI initiation to discontinuation was 79.3 months (range; 22 to 138 months); median duration of CMR before TKI discontinuation was 47.3 months (range; 5 to 97 months). Seven patients showed loss of MMR; 6 relapsed within 6 months and one showed late relapse at 25 months after discontinuation. The cumulative incidence of MMR loss was estimated as 18.8% at 12 months and at 24 months. Fluctuation of BCR-ABL transcript levels below the MMR threshold (〉 two consecutive positive values) was observed in 6.25% of patients at 24 months after ABL TKI discontinuation. Treatment-free remission was estimated as 81.2% at 12 months and at 24 months. The median period of restoration of second CMR was 6.0 months in re-treated patients. No patient died during the follow-up period. TKI-free remission was estimated as 78.1% at 30 months. There was only a significant difference in BCL2L11 (BIM) deletion polymorphism between the patients who maintained and those who lost MMR (p = 0.0253). No significant difference was observed in prior IFNα therapy, time to complete cytogenetic response (CCyR), time to MMR, and time to CMR between relapsing and non-relapsing patients. Conclusion: Our study shows a specific association between BCL2L11 (BIM) deletion polymorphism and clinical outcome after ABL TKI discontinuation in patients with long-lasting molecular undetectable residual disease. BCL2L11 (BIM) deletion polymorphism may predict relapse after ABL TKI discontinuation, which may have an impact on future ABL TKI discontinuation trials. These results further illustrate the importance of single nucleotide polymorphisms in successful long-term treatment of CML. Disclosures Ohyashiki: Bristol-Myers Squibb KK : Research Funding, Speakers Bureau; Novartis KK: Research Funding, Speakers Bureau.

Description: Abstract 531 Hematopoietic stem cells and leukemic stem cells share common features, including self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. Despite these similarities, several stemness factors, such as Hedgehog, Wnt, Notch, and BMI-1 show differential activation in normal versus leukemia stem cells. Hedgehog signaling is increased in BCR-ABL1 positive stem and progenitor cells becoming more active with disease progression. We have previously shown that the combination with the hedgehog signaling pathway inhibitor, vismodegib and ABL tyrosine kinase inhibitor (ABL TKIs) inhibits the minimal residual cells in BCR-ABL1-positive leukemia cells (Blood: ASH Annual Abstracts, Nov 2011; 118: 63). In the present study, we investigated the molecular mechanisms by which vismodegib and LDE225 regulate the self-renewal of primary BCR-ABL1 positive leukemia cells in vivo. To identify the leukemia-propagating cell fraction of BCR-ABL1-positive leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n=1; T315I BCR-ABL1) or Ph-positive acute lymphoblastic leukemia (n=2, T315I BCR-ABL1 and WT-BCR-ABL1) into NOD/SCID/IL-2γc−/− mice. The cell fractions with CD34+CD38−CD19+and CD34+CD38+CD19+ could self-renew and transfer the leukemia in NOD/SCID mice. To investigate the effects of hedgehog inhibition on self-renewal and the relevance of the hedgehog pathway as a therapeutic target in BCR-ABL1 positive leukemia, we examined the activity of vismodegib and LDE225 against CD34+CD38−CD19+, CD34+CD38+CD19+ fractions transferred NOD/SCID mice in vivo. NOD/SCID mice were injected intravenously with BCR-ABL1 positive cells then treated with vismodegib (20 mg/kg; p.o.) or LDE225 (20 mg/kg; p.o.) for 28 days. All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with vismodegib or LDE225 reduced the population of CD34+CD38− positive cells. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of leukemia cells into secondary recipients, subsequently treated with vismodegib or LDE225 for 28 days. Following 30 days, all mice received BCR-ABL1 cells from vehicle treated mice engrafted with leukemia. In contrast, leukemia engraftment was not detected in recipient mice (n=6) from vismodegib or LDE225 treated donors. These results demonstrate the persistent effects of hedgehog inhibition on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of hedgehog pathway modulation on in vitro clonogenic growth. CD34+CD38−CD19+ cells from T315I BCR-ABL1 (n=2) and WT-BCR-ABL1 (n=1) cells were treated with 1 μM of vismodegib or 200 nM of LDE225 for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. Hedgehog pathway inhibition by vismodegib and LDE225 had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by vismodegib and LDE225 (p

Description: Abstract 3745 Introduction: Recently, it has been recognized that some chronic myeloid leukemia (CML) patients with a complete molecular response (CMR) are able to maintain the CMR after discontinuation of imatinib. Mahon et al. reported that among patients with a CMR lasting at least 2 years, CMR was sustained in 41% after discontinuation of imatinib (Lancet Oncol. 2010;11:1029). Similarly, Takahashi et al. reported that 47% of Japanese CML patients with CMR maintained CMR after imatinib discontinuation (Haematologica 2012;97:903). Moreover, Ohyashiki et al. have recently demonstrated that higher peripheral natural killer (NK) cell counts are associated with a reduced risk of relapse after halting imatinib (Br. J. Haematol. 2012;157:254). These findings suggest that, although CML stem cells may remain, even if CMR status is attained, some CML patients could discontinue imatinib therapy and maintain a stable condition, possibly owing to immune surveillance. Aim: To more precisely identify these patients who can safely discontinue imatinib, we characterized the immunophenotype profiles of CML patients. We compared the profiles among CML patients who received imatinib with CMR for more than 2 consecutive years (CMR group), those who could not sustain CMR but maintained a major molecular response (fluctuated CMR group), those who sustained CMR for more than 6 months after discontinuation of imatinib (STIM group), those who relapsed after discontinuation of imatinib (relapse group), and healthy volunteers (control group). Methods: Peripheral blood mononuclear cells (PBMCs) from CML patients and healthy volunteers were separated using a Ficoll density gradient, and immunophenotyping analysis was performed with a 5-color flow cytometry panel, including antibodies against the following cell surface antigens and effector molecules: CD3, CD8, CD45RO, CD56, CCR7, IFN-g, granzyme B, and perforin. After PBMCs were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 4 h in the presence of monensin, cell surface antigens were stained, fixed, and permeabilized. Resultant cells were then intracellularly stained and analyzed with the FACSCanto II flow cytometer. Results: The percentage of effector populations of NK cells, such as interferon (IFN)-g+CD3−CD56+ cells, was significantly higher in the CMR and STIM groups than in the control group. In contrast, the percentage of effector populations of CD8+ T cells, such as IFN-g+CD8+ T cells, was significantly higher in the STIM and control groups than in the CMR group. Moreover, the percentage of effector populations of NK cells, but not CD8+ T cells, was significantly higher in the CMR group than in the fluctuated CMR group. On the other hand, CML patients with a lower percentage of effector populations of NK cells and CD8+T cells, and those with a higher percentage of these effector populations, but whose percentage had decreased after imatinib cessation, had a tendency to relapse after discontinuation of imatinib. Conclusion: The percentage of effector populations of NK cells and CD8+ T cells, such as those expressing IFN-g, correlated highly with sustained CMR after discontinuation of imatinib. Moreover, the activation level of NK cells, but not CD8+ T cells, inversely correlated with the BCR-ABL1 transcript level. Taken together, these results suggest that whether imatinib treatment can be safely discontinued may depend greatly on the immunological activation status of both NK cells and CD8+ T cells in CML patients. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by no efficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). Even in low risk MDS, clonal hematopoiesis already dominates at diagnosis, and clones found in secondary AML originate from the MDS stage of disease, highlighting the need to specifically target the MDS-initiating clone. PF-0449913 is a potent and selective hedgehog pathway inhibitor that act by binding Smoothened (SMO) and blocking signal transduction. In xenograft models of human coloirectal and pancreatic cancer, treatment with PF-04449913 in combination with other anticancer agents reduced the tumor growth. Furthermore, PF-04449913 demonstrated preliminary antitumor activity in a phase I trial, when given as monotherapy in patients with several hematopoietic malignancy. In the present study, we investigated the molecular mechanisms by which PF-04449913 regulate the self-renewal of MDS-derived iPS cells (iPSCs) in vivo. Methods: We generated iPSCs from bone marrow mononuclear cells of two MDS patients (RAEB1 and RAEB2 by WHO clssification) with chromosome 5 deletion and complex karyotypic abnormalities, respectivly. Karyotyping analysis revealed that MDS-derived iPSCs have identical abnormalities to primary MDS cells. We also generated iPSCs from bone marrow mononuclear cells of normal volunteer as control. To investigate the effects of PF-04449913 on self-renewal and the relevance as a therapeutic target in MDS initiating cells, we examined the activity of PF-04449913 against MDS-derived iPSCs transferred NOD/SCID mice in vivo. NOD/SCID mice were injected sucutaneously with MDS-derived iPSCs or normal iPSCs then treated with PF-04449913 (100 mg/kg; p.o.) from day 10 for 28 days. We also used MDS-L, a myelodysplastic cell line establised from MDS patient with del(5q) and complex karyotypic abnormalities for in vitro studies. Results: Both MDS-derived iPSCs transferred NOD/SCID mice and normal iPSCs transferred NOD/SCID mice demonstrated the engraftment of CD34+CD38- positive cells by flow cytometry. However, the treatment with PF-04449913 reduced the population of CD34+CD38-positive cells in MDS-derived iPSCs transferred NOD/SCID mice. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of CD45+ cells into secondary recipients. Following 50 days, all mice treated with vehicle engrafted with CD34+CD38- positive cells. In contrast, CD34+CD38-positive cells engraftment was not detected in recipient mice (n=3) from PF-04449913-treated donors. These results demonstrate the persistent effects of PF-0449913 on long term self-renewing MDS-initiating cells. We further examined the effects of Nanog pathway modulation on in vitro clonogenic growth. CD34+CD38- cells from MDS-derived iPSCs transferred NOD/SCID mice and MDS-L cells were treated with 2 mM of PF-04449913 for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. PF-04449913 had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by PF-04449913 (p