Secondary Colony Formation Research Articles

Abstract A56: TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal

Abstract AML with TP53 mutations (TP53Mut) is a poor-risk AML subtype that is largely insensitive to chemotherapy, targeted agents, and hematopoietic stem cell transplantation. TP53Muts are seen in approximately 10-20% of AML and confer a particularly poor prognosis with 1–2-year survival rates to 0-10%. Self-renewal is the defining feature of leukemia stem cells (LSCs) and is central to leukemia progression and relapse. Mutant p53 protein enhances self-renewal in murine models of AML and in human hematopoietic progenitors. In solid tumors, analysis of a variety of TP53Muts defined the proteasome pathway as a common transcriptional target and mediator of oncogenesis among these mutations. Notably, the proteasome degrades I kappa B which activates NF kappa B, which enforces AML self-renewal. Proteasome inhibitors have shown some efficacy in AML clinical trials but have not been well-evaluated in TP53Mut patients. We hypothesize that the proteasome is mediator of self-renewal in TP53Mut AML. To define the transcriptional features of TP53Mut AML, we analyzed the gene expression data in the BEAT AML dataset of primary human AML samples (Tyner, et al. 2018). Relative to TP53WT (n=377 samples), the TP53 mutant samples (n=36) were enriched in every NF kappa B and proteasome gene-set queried. Next, we analyzed a single cell RNA seq dataset of human AML samples (van Galen et al. 2019) and found that the LSC compartment of TP53Mut samples (n=3) strongly upregulated every proteasome gene set we queried (relative to TP53WT samples (n=13)). A recent in vitro screen identified a panel of small molecule inhibitors that reduced viability in TP53-deleted cell lines (Nechiporuk et al. 2019). We tested the activity of these inhibitors in primary human TP53Mut samples. Four of these inhibitors were effective in reducing colony forming capacity or reduced LSC frequency in TP53Mut samples (n=9). We used CyTOF to assess intracellular signaling pathways in LSCs and found that the inhibitors that were most effective in abolishing colony formation and reducing LSC frequency also attenuated NF kappa B levels in TP53Mut LSCs. Accordingly, we found that knockdown of mutant TP53 reduced NF kappa B levels in Kasumi cells (a TP53R248Q AML cell line). Next, we treated a panel of primary human TP53Mut AML samples with the pan-proteasome inhibitors, carfilzomib and bortezomib (FDA approved for lymphoid malignancies) and an immunoproteasome inhibitor, PR957 (in clinical trials for autoimmune conditions). Each drug independently reduced NF kappa B and led to a significant reduction of primary and secondary colony formation. These data suggest that the proteasome/NF kappa B pathway may be an important determinant of self-renewal function in TP53Mut AML and proteasome inhibitors may target the LSCs of this treatment-refractory AML subtype. Citation Format: Marie Lue Antony, Yoonkyu Lee, Klara E Noble-Orcutt, Zohar Sachs. TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A56.

TMET-30. ENHANCED RADIOSENSITIZATION OF GLIOMAS BY METABOLIC TARGETING OF SALVAGE PATHWAY OF NAD SYNTHESIS THROUGH NAMPT INHIBITION

Abstract BACKGROUND Pathway-specific targeted agents have failed to improve outcome in patients with high grade gliomas. Tumor recurrence is driven by glioma heterogeneity and emergence of therapy-resistant subpopulations. We have previously reported that metabolic targeting of NAMPT, the rate limiting enzyme in the NAD salvage synthesis pathway, circumvents glioma heterogeneity and induces cytotoxicity by mitochondrial membrane depolarization and generation of reactive oxygen species. Based on these data, we examined the potential for NAMPT inhibition to synergize with ionizing radiation to enhance glioma cytotoxicity in vitro and in vivo. METHODS Using a panel of genetically heterogeneous patient-derived glioma stem-like cells (GSC), we examined the effects of NAD targeting using KPT9274, a potent NAMPT inhibitor, in combination with ionizing radiation on cell proliferation and clonogenicity using Cell Titre Glo assay, colony formation assays and secondary sphere formation. Changes in DNA damage-related proteins were visualized by immunofluorescence assay and confocal microscopy. Effects on gene expression and protein levels were assessed using western blot, reverse phase protein array (RPPA) and RNA Seq. RESULTS NAMPT inhibition reduced cell proliferation rate, self-renewal and clonogenic capacity in vitro. Combining KPT-9274 with ionizing radiation caused synergistic inhibition of secondary sphere and colony formation of GSC. RPPA and RNASeq results showed downregulation of several DNA repair proteins which was further confirmed using immunofluorescence staining indicating a crucial role for these proteins in glioma resistance to therapies. Results of ongoing in vivo studies to assess the PK/PD, drug toxicity and survival benefits of this combination therapy in orthotopic GSC intracranial glioma mouse model will be reported. CONCLUSIONS Our results suggest that metabolic targeting through NAMPT inhibition sensitizes glioma cells to radiotherapy. Ongoing studies delineating the mechanistic basis of these effects and their impact in vivo as a potential rationale for clinical assessment will be reported.

ZBP-89 negatively regulates self-renewal of liver cancer stem cells via suppression of Notch1 signaling pathway.

Liver cancer stem cells (LCSCs) initiate hepatocellular carcinoma (HCC) and contribute to its recurrence and treatment resistance. Studies have suggested ZBP-89 as a candidate tumor suppressor in HCC. We explored the role of ZBP-89 in the regulation of LCSCs. This study was performed in liver tissue samples from 104 HCC patients, 2cell lines and mouse tumor models. We demonstrated that ZBP-89 was weakly expressed in LCSCs. Patients with high expression of LCSC markers displayed reduced survivals and higher recurrence rates after curative surgical operation. The expression of ZBP-89 was predictive for decreased recurrence. LCSC markers were negatively correlated with ZBP-89 in HCC tissues and in enriched liver tumor spheres. The exogenous expression of ZBP-89 attenuated the tumor-sphere formation and secondary colony formation capabilities of LCSCs in vitro and tumorigenicity in vivo. Furthermore, the negative effect of ZBP-89 on cancer stemness was Notch1-dependent. Localized with Notch1 intracellular domain (NICD1) in the nucleus, ZBP-89 repressed the Notch1 signaling pathway by competitive binding to NICD1 with MAML1. Collectively, ZBP-89 negatively regulates HCC stemness via inhibiting the Notch1 signaling.

The long non-coding RNA Snhg3 is essential for mouse embryonic stem cell self-renewal and pluripotency

BackgroundSmall nucleolar RNA host gene 3 (Snhg3) is a long non-coding RNA (lncRNA) that was shown to participate in the tumorigenesis of certain cancers. However, little is known about its role in embryonic stem cells (ESCs).MethodsHere, we investigated the role of Snhg3 in mouse ESCs (mESCs) through both loss-of-function (knockdown) and gain-of-function (overexpression) approaches. Alkaline phosphatase staining, secondary colony formation, propidium iodide staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to access self-renewal capacity, whereas immunofluorescence, qRT-PCR, and embryoid body formation were performed to examine pluripotency. In addition, the effect of Snhg3 on mouse embryonic development was determined based on the morphological changes, blastocyst rate, and altered pluripotency marker (Nanog, Oct4) expression. Moreover, the relationship between Snhg3 and key pluripotency factors was evaluated by chromatin immunoprecipitation qPCR, qRT-PCR, subcellular fractionation, and RNA immunoprecipitation. Finally, RNA pull-down and mass spectrometry were applied to explore the potential interacting proteins of Snhg3 in mESCs.ResultsWe demonstrated that Snhg3 is essential for self-renewal and pluripotency maintenance in mESCs. In addition, Snhg3 knockdown disrupted mouse early embryo development. Mechanistically, Snhg3 formed a positive feedback network with Nanog and Oct4, and 126 Snhg3-interacting proteins were identified in mESCs.ConclusionsSnhg3 is essential for mESC self-renewal and pluripotency, as well as mouse early embryo development.

Abstract 5889: Cancer associated fibroblasts regulate cancer stem cell functions in pancreatic ductal adenocarcinoma

Abstract Introduction: Despite recent advancements in treatment, the outcome of pancreatic ductal adenocarcinoma (PDAC) remains dismal due to high relapse rate and metastatic dissemination. PDAC cancer stem cells (CSCs) have been shown to play a role in these processes and are associated with worse median survival in patients. Several cell-intrinsic mechanisms of CSC regulation have been identified in PDAC, however the role of the altered tumor microenvironment remains largely unknown. We focusd on the impact of cancer associated fibroblasts (CAFs) on CSCs properties in PDAC. Methods: Stromal fibroblast lines were established from surgically resected PDAC tissues and were marked by specific fluorophore. Following co-culture with CAFs, tumor cells from PDAC cell lines or low passage xenografts derived from primary surgical specimens cells were isolated and evaluated for colony formation and migration. The effect of Focal Adhesion Kinase (FAK) on CAF-mediated clonogenic growth was evaluated using the small molecule FAK inhibitor PF573228 or overexpression of full-length of FAK in PDAC cells. Results: Following co-culture with CAFs, the clonogenic growth of PDAC cell lines increased by 1.3-2.5 fold compared to cells incubated alone (p ≤ 0.05). Furthermore, CAFs enhanced colony formation of cells from patient derived xenografts by at least 4 fold (p ≤ 0.001). To examine the effect of CAFs on PDAC self-renewal, tumor colonies were harvested and replated in methylcellulose. Although cells were not further exposed to CAFs, secondary colony formation of cells originally co-cultured with CAFs increased by > 1.3 fold. We also found that the frequency of ALDH+ PDAC CSCs increased by 12-19 fold compared to control cells. CSCs are also highly migratory and express factors suggestive of the epithelial to mesenchymal transition (EMT) suggesting that they play an important role in metastatic disease. Following co-culture, the migration of PDAC cells increased by 1.2-2.1 fold (p ≤ 0.05) with increased expression of the EMT associated genes SNAIL1, SNAIL2, ZEB1, ZEB2, N-cadherin, and Vimentin. PDAC cells induced CAF activation as evidenced by enhanced αSMA expression. Since activated CAFs are known to secret extracellular matrix proteins, we examined the activation of FAK downstream of integrin signaling and found increased phosphorylation of FAK at Y397. To determine the importance of FAK activation on the interaction between CAFs and PDAC cells, cells were treated with the FAK inhibitor PF573228 that significantly inhibited tumor colony formation. In contrast, the overexpression of FAK-FL further increased CAF-mediated PDAC clonogenic growth. Conclusion: CAFs enhance PDAC CSCs including clonogenic growth, migration, and self-renewal. These effects are partially mediated by FAK, and FAK inhibitors may represent a novel strategy to modulate the interaction of PDAC cells and the tumor microenviroment. Citation Format: Asma Begum, Ross McMillan, Yu-tai Chang, Vesselin Penchev, NV Rajeshkumar, Anirban Maitra, Michael G. Goggins, James R. Eshelman, Christopher Wolfgang, Zeshaan A. Rasheed, William H. Matsui. Cancer associated fibroblasts regulate cancer stem cell functions in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5889. doi:10.1158/1538-7445.AM2017-5889

Internal tandem duplication of FLT3 deregulates proliferation and differentiation and confers resistance to the FLT3 inhibitor AC220 by Up-regulating RUNX1 expression in hematopoietic cells

Internal tandem duplication in the FLT3 gene (FLT3/ITD), which is found in patients with acute myeloid leukemia (AML), causes resistance to FLT3 inhibitors. We found that RUNX1, a transcription factor that regulates normal hematopoiesis, is up-regulated in patients with FLT3/ITD(+) AML. While RUNX1 can function as a tumor suppressor, recent data have shown that RUNX1 is required for AML cell survival. In the present study, we investigated the functional role of RUNX1 in FLT3/ITD signaling. FLT3/ITD induced growth factor-independent proliferation and impaired G-CSF mediated myeloid differentiation in 32D hematopoietic cells, coincident with up-regulation of RUNX1 expression. Silencing of RUNX1 expression significantly decreased proliferation and secondary colony formation, and partially abrogated the impaired myeloid differentiation of FLT3/ITD(+) 32D cells. Although the number of FLT3/ITD(+) 32D cells declined after incubation with the FLT3/ITD inhibitor AC220, the cells became refractory to AC220, concomitant with up-regulation of RUNX1. Silencing of RUNX1 abrogated the emergence and proliferation of AC220-resistant FLT3/ITD(+) 32D cells in the presence of AC220. Our data indicate that FLT3/ITD deregulates cell proliferation and differentiation and confers resistance to AC220 by up-regulating RUNX1 expression. These findings suggest an oncogenic role for RUNX1 in FLT3/ITD(+) cells and that inhibition of RUNX1 function represents a potential therapeutic strategy in patients with refractory FLT3/ITD(+) AML.

MEK-ERK Activity Regulates the Proliferative Activity of Fetal Hepatoblasts Through Accumulation of p16/19(cdkn2a).

Hepatoblasts are somatic progenitor cells in the fetal liver, which retain a high proliferative capacity and differentiate into both hepatocytes and cholangiocytes in vivo. Although efficient expansion of hepatoblasts in vitro has been difficult without genetic modification, we have previously demonstrated that the interaction with mesenchymal cells is important for expansion of hepatoblasts in vitro. In this study, we show cell signaling pathways regulating the long-term proliferative ability of hepatoblasts. Individual primary hepatoblasts derived from mouse fetal livers formed large colonies when cocultured with mesenchymal feeder cells; however, secondary colony formation was unsuccessful, indicating that in vitro culture could induce short-term, but not long-term, proliferation. When the MEK inhibitor, PD0325901, was added to these cultures, hepatoblasts formed large colonies containing many Ki-67-positive cells. Expression of p16/19(cdkn2a), a cyclin-dependent kinase inhibitor, was induced after 3-6 days culture of hepatoblasts, whereas PD0325901 significantly suppressed this expression. Consistent with these observations, fetal hepatoblasts derived from p16/19(cdkn2a) knockout mice showed long-term proliferation without PD0325901, suggesting that MEK activity induced cell cycle arrest through accumulation of p16/19(cdkn2a). In transplantation assays, we could demonstrate that in vitro expanded hepatoblasts could proliferate and differentiate into hepatocytic and cholangiocytic cells in injured livers. It should also be noted that ERK in primary hepatoblasts was not highly activated during fetal liver development. Collectively, all these findings suggest that the MEK/ERK-independent pathway in the fetal liver is involved in hepatoblast proliferation to avoid accumulation of cyclin-dependent kinase inhibitor.

Existence, Functional Impairment, and Lung Repair Potential of Endothelial Colony-Forming Cells in Oxygen-Induced Arrested Alveolar Growth

Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth. Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth-arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood-derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension. Impaired ECFC function may contribute to arrested alveolar growth. Cord blood-derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage.

A branching process model for the analysis of abortive colony size distributions in carbon ion-irradiated normal human fibroblasts

A single cell can form a colony, and ionizing irradiation has long been known to reduce such a cellular clonogenic potential. Analysis of abortive colonies unable to continue to grow should provide important information on the reproductive cell death (RCD) following irradiation. Our previous analysis with a branching process model showed that the RCD in normal human fibroblasts can persist over 16 generations following irradiation with low linear energy transfer (LET) γ-rays. Here we further set out to evaluate the RCD persistency in abortive colonies arising from normal human fibroblasts exposed to high-LET carbon ions (18.3 MeV/u, 108 keV/µm). We found that the abortive colony size distribution determined by biological experiments follows a linear relationship on the log–log plot, and that the Monte Carlo simulation using the RCD probability estimated from such a linear relationship well simulates the experimentally determined surviving fraction and the relative biological effectiveness (RBE). We identified the short-term phase and long-term phase for the persistent RCD following carbon-ion irradiation, which were similar to those previously identified following γ-irradiation. Taken together, our results suggest that subsequent secondary or tertiary colony formation would be invaluable for understanding the long-lasting RCD. All together, our framework for analysis with a branching process model and a colony formation assay is applicable to determination of cellular responses to low- and high-LET radiation, and suggests that the long-lasting RCD is a pivotal determinant of the surviving fraction and the RBE.

Replication Factor C3 Is a Direct Target Of CREB, Promotes G1/S Transition Of Acute Myeloid Leukemia Cells, and Increases Hematopoietic Stem/Progenitor Cell Self-Renewal

CREB (cAMP Response Element Binding protein) promotes cellular transformation of hematopoietic cells and proliferation of myeloid leukemia cells. However, the underlying mechanisms of CREB function in leukemic transformation and hematopoiesis are not fully understood. To address this, we have investigated the downstream pathways of CREB activation in proliferation using a human acute myeloid leukemia (AML) cell line KG1 cells knocked-down for CREB with specific shRNAs. The CREB-knockdown KG1 cells were significantly defective in proliferative capability compared to control cells [cell number after 4d (X105), seeding (1X105), control vs. CREB-knockdown: 34.18 +/– 1.27 vs. 14.52 +/– 0.46, n=3, p< 0.01, mean +/– SEM]. In order to characterize the specific role of CREB in cell proliferation, we analyzed cell cycle progression patterns of CREB-knockdown and control KG1 cells after release from mitotic arrest. Our results indicated that G1 to S phase transition as assessed by % S phase was impeded by CREB-knockdown [S phase (%), control vs. CREB-knockdown cells, 8h after release: 53.29 +/– 0.54 vs. 23.57 +/– 1.69; 12h: 66.92 +/– 0.63 vs. 45.16 +/– 0.50, n=3, p< 0.01, mean +/– SEM]. To identify potential CREB target genes, we chose several cell cycle related genes such as CCNE1, CCNA1, CCNB1 and PCNA and compared their RNA expression levels in the CREB-knockdown with those in control KG1 cells after release from mitotic arrest. To our surprise, we failed to detect any noticeable differences in the mRNA expression levels of those genes between CREB-knockdown and control KG1 cells. In an effort to search for CREB responsive target genes, we analyzed additional CREB targets previously identified from microarray data (Pellegrini et al BMC Cancer 2008). We found that expression of replication factor C3 (RFC3), a 38kDa subunit of the RFC complex involved in DNA replication and repair processes, was significantly reduced in CREB-knockdown cells compared to control cells [38 +/– 1% of control, n=3, p<0.01]. CREB-knockdown also inhibited RFC3 mRNA expression in U937 and HL60 AML cell lines. Consistent with these results, mRNA expression levels of RFC3 appeared to be closely correlated with those of CREB when we examined bone marrow samples obtained from AML patients [n = 16, Pearson coefficient = 0.6366, p = 0.0008]. Moreover, we found that CREB directly interacted with the CRE site in the RFC3 promoter region in vivo, as assessed by chromatin immunoprecipitation assays. Exogenous overexpression of RFC3 in CREB-knockdown KG1 cells restored the defective G1/S progression [S phase (%), CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 9h after release: 38.97 +/– 0.45 vs. 62.24 +/– 1.06; 12h: 48.12 +/– 0.60 vs. 67.70 +/– 1.15, n=3, p< 0.01, mean +/– SEM]. Taken together, these results suggest that RFC3 may act as a novel downstream oncogenic target of activated CREB in AML cells.We previously reported that CREB is a critical regulator of normal myelopoiesis (Cheng et al Blood 2008). To determine whether RFC3 could exert similar effects on normal hematopoiesis, we compared human umbilical cord blood derived CD34-positive cells with and without RFC3 overexpression for the capacity to form hematopoietic colonies. Overexpression of RFC3 in the CD34-positive cells resulted in significant increases of multi-potential CFU-GEMM colony numbers [without vs. with overexpression of RFC3 (per 1000 cells): 3.2 +/– 1.3 vs. 22.3 +/– 3.3, n=3, p< 0.01, mean +/– SEM]. The RFC3 effect on stimulating colony formation was magnified in secondary colony forming assays [without vs. with overexpression of RFC3 (per 100,000 cells): 10.7 +/– 3.5 vs. 180.2 +/– 44.4, n=3, p< 0.05, mean +/– SEM]. Since the formation of secondary colonies was derived mainly from residual stem/progenitor cell populations after long-term culture, RFC3 overexpression may enhance self-renewal of stem/progenitor cells. In conclusion, our results suggest that RFC3 is able to promote G1/S transition in a human AML cell line downstream of CREB activation. In addition, we provide evidence that RFC3 is involved in normal hematopoiesis and contributes to increased self-renewal potential of hematopoietic stem/progenitor cells. Our data demonstrate that RFC3 plays multiple roles in promoting AML cells proliferation as well as normal myelopoiesis through increasing the self-renewal potential of hematopoietic stem/progenitor cells in response to CREB activation. Disclosures:No relevant conflicts of interest to declare.

Order Matters: Sequence Of Mutation Acquisition In Myeloproliferative Neoplasms Impacts Disease Pathogenesis and Stem Cell Potency

Cancers evolve as a consequence of the stepwise accumulation of somatic lesions, with competition between subclones and sequential subclonal evolution. Some driver mutations arise either early or late in the evolution of different individual tumors, indicating that the final malignant properties of a subclone reflect the sum of mutations acquired rather than the order in which they arose. However virtually nothing is known about the cellular consequences of altering the order in which mutations are acquired.The myeloproliferative neoplasms (MPNs) readily permit clonal analysis and are chronic malignancies, thereby facilitating the dissection of disease evolution and intra-tumoral clonal architecture. In this study, we genotyped >7000 colonies from 24 MPN patients who harbored mutations in both JAK2 and TET2. We found that mutation of JAK2 and TET2 each occurred first in 12/24 patients, with TET2-first and JAK2-first patients observed in all 3 MPN subtypes. Patients who acquired a TET2 mutation first presented on average 12.3 years later than JAK2-first patients (p=0.0043) and had a lower proportion of JAK2V617F homozygous erythroid colonies (p=0.0001) suggesting that the order of acquisition affected both cellular composition and disease evolution. Moreover, compared to TET2-first patients, JAK2-first patients had an increased frequency of megakaryocyte/erythrocyte progenitors (p=0.0001) and decreased frequency of common myeloid progenitors (p=0.001). In order to determine whether disease evolution was also affected at the stem cell level, we isolated individual HSCs (lin-CD34+CD38+CD90-CD45RA+) and found that JAK2 single-mutant HSCs were significantly less prevalent than their JAK2/TET2 double-mutant counterparts. In marked contrast, TET2 single-mutant HSCs were significantly more prevalent than JAK2/TET2 double-mutant HSCs, suggesting that TET2 single-mutant HSCs exhibit a substantial self-renewal advantage and were not out-competed by their double-mutant progeny. Consistent with this interpretation, functional progenitor expansion from single cell cultures of individual HSCs (as assessed by secondary colony formation) was differentially altered by the second mutation acquired. Whereas the mean number of secondary colonies produced by double-mutant HSCs was increased when TET2 was the second mutation (p=0.012), the mean number of colonies was decreased when JAK2 was the second mutation (p=0.002) despite both HSCs bearing the same set of genetic lesions.Together our data indicate that acquisition of a JAK2 mutation reduces the competitiveness of TET2 single mutant HSCs, whereas acquisition of a TET2 mutation enhances that of JAK2 single mutant HSCs. These observations represent the first demonstration that the order of mutation acquisition influences stem and progenitor cell behaviour and clonal evolution in cancer. Disclosures:No relevant conflicts of interest to declare.

Flt3/ITD Blocks Myeloid Differentiation Of Hematopoietic Cells By Up-Regulating Runx1

Internal-Tandem-Duplication mutations in the FLT3 (FLT3/ITD) gene are detected in 30% of patients with acute myeloid leukemia (AML) and are associated with extremely poor prognoses. The lack of significant efficacy of FLT3/ITD inhibitors underscores the need to identify FLT3/ITD-specific signaling pathways that are distinct from those that occur in normal hematopoietic cells to develop novel therapeutic approaches. FLT3/ITD is classified as a “class I mutation” that drives the proliferation of leukemia cells. In addition to mutation of FLT3/ITD, a “class II mutation” that blocks differentiation of the pre-leukemic clone is generally required for the development of AML. For instance, dominant negative mutations of RUNX1 are occasionally found in patients with AML. These mutations of RUNX1 cause AML by blocking the differentiation of leukemia cells in combination with the mutation of FLT3/ITD. RUNX1 is a core-binding transcription factor and plays an important role in hematopoietic homeostasis, particularly differentiation and proliferation. Loss of RUNX1 blocks hematopoietic differentiation and is associated with the emergence of a primitive hematopoietic compartment, suggesting that RUNX1 generally induces differentiation of hematopoietic cells. However, the functional role of RUNX1 as a down-stream effector of FLT3/ITD has not been characterized. Herein, we investigated the role of Runx1 in aberrant proliferation and differentiation of hematopoietic cells induced by Flt3 /ITD.A comparison of RUNX1 expression levels in AML patients for whom information has been deposited in the public gene expression profile database (GSE1159) revealed that RUNX1 mRNA expression was significantly higher in FLT3/ITD+AML cells (N=78) than in FLT3/ITD-AML cells (N=190, P<0.05). The mRNA microarray analysis consistently demonstrated that Runx1 is up-regulated by Flt3/ITD in Ba/F3 cells. Up-regulation of Runx1 by Flt3/ITD was validated in Ba/F3 cells and 32D cells by quantitative RT-PCR. Incubation of control 32D cells with 20 ng/ml of G-CSF increased the number of Gr-1+/Mac-1+cells, whereas the induction of myeloid differentiation by G-CSF was abrogated by the overexpression of Flt3/ITD in 32D cells. By contrast, transduction of shRNA specific for Runx1 into Flt3/ITD+32D cells inhibited the expression of Runx1 mRNA by 60 % but increased the number and the proportion of Gr-1+/Mac-1+cells ; these effects were enhanced by incubation with G-CSF. These data indicate that Runx1 mediates the block of differentiation toward the myeloid lineage that is induced by Flt3/ITD. Moreover, the number of colony-forming units (CFU) over-expressing Flt3/ITD cultured in the absence of growth factors was reduced by Runx1-shRNA without affecting the total cell number in the suspension culture, as compared to Flt3/ITD+32D cells transduced with control-shRNA. This implies that antagonizing Runx1 facilitates the production of terminally differentiated cells that have lost colony-forming ability, thereby reducing the CFU number without altering the total number of cells. Finally, Runx1-shRNA inhibited the formation of secondary CFU colonies derived from the primary Flt3-ITD-over-expressing CFU colonies.Our results suggest that Flt3/ITD blocks myeloid differentiation of Flt3/ITD+cells by up-regulating Runx1 expression. The blocking of differentiation mediated by Runx1 in Flt3/ITD+cells is in contrast to the cell differentiation-inducing role of Runx1 in normal hematopoiesis, suggesting that the function of Runx1 in Flt3/ITD+cells may be distinct from that in normal cells. The reduction of secondary CFU colonies by Runx1-shRNA suggests that Runx1 may mediate self-renewal of Flt3/ITD+hematopoietic progenitor cells. These findings suggest that antagonizing RUNX1 may represent a novel therapeutic strategy to induce terminal differentiation of FLT3/ITD+AML cells in AML patients, in addition to inhibiting their aberrant proliferation. Disclosures:No relevant conflicts of interest to declare.

Ras-Pathway Inhibition With Targeted Therapies Abrogates Self-Renewal In Acute Myelogenous Leukemia

Hyperactivated Ras-pathways serve as oncogenic drivers in multiple human tumors including acute myelogenous leukemia (AML) (Ahearn et al. Nat Rev Mol Cell Biol 2011). The specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Recently, we have shown that NRASG12V–activated signaling pathways are critical to leukemia stem cell maintenance (Sachs et al. submitted). To elucidate which Ras-activated signaling molecules mediate self-renewal in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V) and develops AML (Kim et al. Blood 2009). Primary leukemia cells were treated with therapeutic agents targeting Ras-activated signaling pathways. We used PD325901 to inhibit the Mek-Erk pathway, GDC0941 to inhibit the Pi3k pathway, and RAD001 to inhibit the mTor pathway. Using MTS assays, we identified the IC50 dose for each of these agents. Inhibitor-treated leukemia cells were submitted for RNA sequencing in order to investigate the effects of these agents on leukemia gene expression. Previously, we identified a list of NRASG12V responsive genes in our model. In these studies, we identified that PD325901-treatment most closely recapitulates the effect of NRASG12V inhibition on this comprehensive list of RAS-responsive genes. However, when we study the effects of these inhibitors on the subset of RAS-responsive genes that mediate leukemia self-renewal, we find that both PD325901 and RAD001 independently recapitulate the effects of NRASG12V withdrawal on this subset of genes implicating the Mek and mTor pathways in leukemia self renewal. Next, we treated primary leukemia cells with the IC50 dose of each drug and plated them in colony forming assays. We found that Mek or mTor inhibition, but not Pi3k inhibition, abrogated secondary colony formation corroborating our gene expression analyses and showing that, at doses that have equivalent effects on cell growth, only the Mek and mTor pathways are important for leukemia cell stem cell maintenance. These studies provide potential targets for leukemia stem cell-specific therapies. Disclosures:Sachs:Silicon Valley Biosystems: Consultancy. Bendall:DVS Sciences: Consultancy. Nolan:SAB for DVS Sciences and Nodality: Chairman Other; Cell Signalling Technologies and Becton Dickenson, Inc: Consultancy. Largaespada:Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.

Molecular Determinants of Bendamustine (BDM) Toxicity towards Hodgkin (H) and Reed-Sternberg (RS) Cell Lines From Hodgkin Lymphoma (HL).

AbstractAbstract 2763Patients with HL recurring after stem cell transplantation (SCT) are mostly incurable. Therefore development of new agents and strategies is an impellent medical need in this setting. BDM is a hybrid purine analogue/bi-functional alkylator active in B-cell tumors. Despite preliminary evidences of efficacy in refractory HL, the molecular mechanisms underlying the potential activity of BDM towards malignant H-RS cells were never explored.We investigated patterns of BDM cytotoxicity (12.5 to 100 mmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its time-dependent (8, 24, 48, 72 hrs) effects on genes involved in DNA-damage stress and repair response, apoptosis and cell cycle checkpoints by Q-RT-PCR. BDM induced a significant time- and dose-dependent inhibition of growth and survival in all cell types. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 10.7 mmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 11.1, 12.4, 14.8 and 16.2 mmol/L, respectively. BDM elicited a dose-dependent increase of apoptosis (30% to 50% at 72 hrs) in all cell lines, as shown by Annexin-V/propidium iodide staining. The exquisite sensitivity to BDM of L1236 cells was confirmed by clonogenic assays, since these cells, after a 24 hr exposure, showed the lowest IC50 for secondary colony formation (0.7± 0.06 mmol/L) as compared to all other cell lines (IC50 range: 3.1 ± 0.28 to 15.0 ± 1.27 mmol/L).Most notably, however, BDM, within the same concentration range, activated different cell death subroutines among the various cell lines. Q-RT-PCR disclosed that BDM-induced cell death in L1236 was mainly dependent on triggering of apoptosis, as shown by the early (8–24 hrs) up regulation of the proapoptotic genes NOXA and p21, but not p27, without appreciable changes in expression levels of genes related to activation of the mitotic catastrophe process, i.e. PLK1, Aurora A kinase (AAK), and cyclin B1. In contrast, induction of mitotic catastrophe was a main determinant of BDM action on KMH2, L428 and L540 cells, as shown by early (8 hrs) and sustained (48 hrs) down-regulation of PLK1, AAK and cyclin B1 genes, without early changes in NOXA and p21 expression levels. This was confirmed by highly aberrant mitosis and further multinucleation. Interestingly, BDM induced the sequential activation of both these cell death pathways in HDLM2 cells only. In this cells, the early (8–24 hrs) down regulation of PLK1, AAK and cyclin B1 genes, was later (48–72 hrs) followed by a significant induction of NOXA, p21 and p27 genes. The highest sensitivity of L1236 cells to BDM correlated with the delayed (72 hrs) induction of the DNA-repair genes EXO-1 and ATR, but not ATM, as opposed to the earlier (24 hrs) and sustained up regulation of these DNA-repair genes, and of ATM, in all other cell types. Accordingly, only after 72 hrs from exposure to BDM, the proliferation-related genes C-MYC, E2F2 and cyclins (D1, D2) were up regulated in surviving L1236 cells, along with a >70% reduction in G0/G1 cells, a significant (48%) increase of cells in the G2 phase of the cell cycle and a 20% increment of those in S phase. Conversely, in the other cell lines, S phase accumulation (35% to 60.5% increase) and up regulation of proliferation-related genes in surviving cells occurred, as early as 24 hrs after exposure to BDM.Overall, these results indicate that BDM affects different cell death pathways among HL-derived cell lines. Specifically, in L1236 cells the agent induces the early up regulation of proapototic genes and G2 arrest, accompanied by a delayed activation of DNA repair genes. This changes may lead to cell death through apoptosis rather than mitotic catastrophe which, conversely, appeared the predominant cell death pathway activated by BDM in the other cell lines. Notably, while determinants of BDM toxicity in ‘bona fide' HL cell lines consistently overlap with those described for tumor B-cells of non-Hodgkin lymphomas (mitotic catastrophe), L1236 cells display a different response pattern to the agent, more reminiscent of BDM action on myeloma tumor cells. Since only L1236 cells are clonotypically related to primary H-RS cells from which were derived, our results suggest that optimal BDM dosing and scheduling in HL may be different from those adopted for other B-cell tumors. In this sense, a lower BDM dosing through a weekly schedule is currently being tested at our institution in refractory HL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Regulation of hematopoietic stem cells using protein transduction domain–fused Polycomb

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

Holoclone Forming Cells from Pancreatic Cancer Cells Enrich Tumor Initiating Cells and Represent a Novel Model for Study of Cancer Stem Cells

BackgroundPancreatic cancer is one of the direct causes of cancer-related death. High level of chemoresistance is one of the major obstacles of clinical treatment. In recent years, cancer stem cells have been widely identified and indicated as the origin of chemoresistance in multi-types of solid tumors. Increasing evidences suggest that cancer stem cells reside in the cells capable of forming holoclones continuously. However, in pancreatic cancer, holoclone-forming cells have not been characterized yet. Therefore, the goal of our present study was to indentify the holoclone-forming pancreatic cancer stem cells and develop an in vitro continuous colony formation system, which will greatly facilitate the study of pancreatic cancer stem cells.Methodology/Principal FindingsPancreatic cancer cell line BxPC3 was submitted to monoclonal cultivation to generate colonies. Based on the morphologies, colonies were classified and analyzed for their capacities of secondary colony formation, long-term survival in vitro, tumor formation in vivo, and drug resistance. Flowcytometry and quantitative RT-PCR were performed to detect the expression level of cancer stem cells associated cell surface markers, regulatory genes and microRNAs in distinct types of colonies. Three types of colonies with distinct morphologies were identified and termed as holo-, mero-, and paraclones, in which only holoclones generated descendant colonies of all three types in further passages. Compared to mero- and paraclones, holoclones possessed higher capacities of long-term survival, tumor initiation, and chemoresistance. The preferential expression of cancer stem cells related marker (CXCR4), regulatory genes (BMI1, GLI1, and GLI2) and microRNAs (miR-214, miR-21, miR-221, miR-222 and miR-155) in holoclones were also highlighted.Conclusions/SignificanceOur results indicate that the pancreatic tumor-initiating cells with high level of chemoresistance were enriched in holoclones derived from BxPC3 cell line. Generation of holoclones can serve as a novel model for studying cancer stem cells, and attribute to developing new anti-cancer drugs.

Effect of bendamustine on apoptosis and colony-initiating precursors in Hodgkin lymphoma cells.

e18559 Background: Hodgkin lymphoma (HL) recurring after stem cell transplantation is incurable and active agents are needed in this setting. Bendamustine is a bi-functional alkylator active in non-Hodgkin lymphoma. Activity of bendamustine towards tumor cells of HL has never been previously described. Methods: A panel of human cell lines (L1236, L428, KMH2, HDLM2, L540), validated models for HL, was studied. Cells were exposed to increasing concentrations of bendamustine (12.5 to 100 µmol/L) and analyzed for proliferation, apoptosis and cell cycle changes. Effect of bendamustine on HL clonogenic precursors was assessed by primary/secondary methylcellulose assays and colony cells characterization (CD30, aldehyde dehydrogenase [ALDH] or Hoechst [Hcst]-33342). Results: Bendamustine displayed a significant time- and dose-dependent antiproliferative effect on all HL cell lines, with an observed IC50 between 25-50 µmol/L at 48 hrs. In all lines, at least 50% of cells showed Annexin-V binding following 24 hrs of exposure to concentrations lower than 100 µmol/L, with 70% to 90% of apoptotic cells at longer times (48-72 hrs). Sensitivity to Bendamustine of HL cells was independent from the presence of p53 mutations. In all HL lines, exposure to a concentration as low as 25 µmol/L induced >50% of cells to accumulate in the G2/M phase of the cell cycle with presence of tetraploid nuclei witnessing mitotic defects. Finally, a 24 hr-exposure to very low concentrations (IC50: 17-38 µmol/L) of bendamustine was able to inhibit primary colony formation in all HL cell lines irrespective of p53 status. Intriguingly, a significantly lower concentration of the agent (IC50: 3.1-15.0 µmol/L) was able to suppress formation of secondary colonies, which were sustained, in L428 and KMH2 cells, by CD30+/ALDH)hi or Hcsthi precursor cells. Conclusions: Bendamustine displays marked antiproliferative and pro-apototic effects on cultured HL cells, irrespective of p53 status, by promoting G2/M arrest and changes compatible with mitotic catastrophe. HL colony-initiating precursors are highly sensitive to extremely low concentrations of the agent. Clinical testing of bendamustine in HL both as single agent and in combination is warranted.

Epigenetic Gene Silencing is a Novel Mechanism Involved in Delayed Manifestation of Radiation-Induced Genomic Instability in Mammalian Cells

We examined mechanisms involved in delayed mutagenesis in CHO-LacZeo cells harboring the fusion gene between the bacterial LacZ and the Zeocin-resistance genes. After X irradiation, Zeocin-resistant primary colonies were isolated, and the primary clones were subjected to the secondary colony formation in the absence of Zeocin. We found that the surviving primary clones showed a significantly higher delayed mutation frequency compared with those derived from nonirradiated CHO-LacZeo cells. The mutation spectrum of the LacZ gene was analyzed by the LacZ gene-specific PCR. We found that more than 90% of the spontaneous and direct mutants were PCR-product negative, indicating that deletion of the LacZ gene was a predominant change in these mutants. While deletion of the LacZ gene was also observed in delayed mutants, we found that more than 20% of delayed mutants had a PCR product similar to that of the parental CHO-LacZeo cells. These PCR product-positive mutants spontaneously reverted to LacZ-positive (LacZ(+)) cells, and all of these mutants became LacZ-positive after 5-azacytidine treatment. These results indicate that epigenetic gene silencing, in addition to elevated recombination, is involved in delayed mutagenesis, which is a novel mechanism underlying delayed manifestations of radiation-induced genomic instability.

Inhibition of Chronic Myeloid Leukemia Stem Cells by the Combination of the Hedgehog Pathway Inhibitor LDE225 with Nilotinib

AbstractAbstract 514 Background:Tyrosine kinase inhibitors (TKI), although effective in inducing remissions and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse on stopping treatment. Additional strategies to enhance elimination of LSC in TKI-treated CML patients are required. The Hedgehog (Hh) pathway, important for developmental hematopoiesis, has been shown to be activated in BCR-ABL-expressing LSC, in association with upregulation of Smoothened (SMO), and contributes to maintenance of BCR-ABL+ LSC. However the role of Hh signaling in chronic phase (CP) CML LSC is not clear. LDE225 (LDE, Novartis Pharma) is a small molecule SMO antagonist which is being clinically evaluated in patients with solid tumors. We have reported that LDE does not significantly affect proliferation and apoptosis of primary CP CML CD34+ cells, or reduce colony growth in CFC assays, but results in significant reduction in CML CFC replating efficiency and secondary colony formation. Treatment with LDE + Nilotinib resulted in significant reduction in colony formation from CD34+ CML cells in LTCIC assays compared to Nilotinib alone or untreated controls. These observations suggest that LDE may preferentially inhibit growth of primitive CML progenitors and progenitor self-renewal. We therefore further investigated the effect of LDE on growth of primitive CML LSC in vivo. Methods and Results:1) CP CML CD34+ cells were treated with LDE (10nM), Nilotinib (5μ M) or LDE + Nilotinib for 72 hours followed by transplantation into NOD-SCID γ-chain- (NSG) mice. Treatment with LDE + Nilotinib resulted in reduced engraftment of CML CD45+ cells (p=0.06) and CD34+ cells (p=0.02) compared with controls, and significantly reduced engraftment of CML cells with CFC capacity (p=0.005). In contrast LDE or Nilotinib alone did not reduce CML cell engraftment in the bone marrow (BM) compared with untreated controls. LDE, Nilotinib, or LDE + Nilotinib treatment did not significantly inhibit engraftment of normal human CD34+ cells in NSG mice compared to controls. 2) We also used the transgenic Scl-tTa-BCR-ABL mouse model of CP CML to investigate the effect of in vivo treatment with LDE on CML LSC. BM cells from GFP-SCL-tTA/BCR-ABL mice were transplanted into wild type congenic recipients to establish a cohort of mice with CML-like disease. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Transplanted CML cells were identifiable through GFP expression. Mice were treated with LDE225 (80mg/kg/d by gavage), Nilotinib (50 mg/kg/d by gavage), LDE + Nilotinib, or vehicle alone (control) for 3 weeks. Treatment with Nilotinib, LDE, and LDE + Nilotinib resulted in normalization of WBC and neutrophil counts in peripheral blood. LDE + Nilotinib treatment significantly reduced the number of splenic long term hematopoietic stem cells (LT-HSC, Lin-Sca-1+Kit+Flt3-CD150+CD48-, p<0.01) and granulocyte-macrophage progenitors (GMP) compared to controls, but did not significantly alter LT-HSC numbers in the BM. LDE alone reduced splenic LT-HSC but not GMP, whereas Nilotinib alone did not reduce LT-HSC numbers in spleen or BM but significantly reduced splenic GMP numbers. The mechanisms underlying enhanced targeting of LSC in the spleen compared to the BM are not clear but could reflect greater dependence on Hh signaling in the context of the splenic microenvironment and/or relocalization of LDE treated LT-HSC to BM. Experiments in which BM and spleen cells from treated mice were transplanted into secondary recipients to determine functional stem cell capacity of remaining LT-HSC are ongoing. Importantly mice treated with LDE + Nilotinib demonstrated enhanced survival on follow up after discontinuation of treatment compared with control mice or mice treated with LDE or Nilotinib alone. Conclusions:We conclude that LDE225 can target LSC from CP CML patients and in a transgenic BCR-ABL model of CP CML, and that LDE + Nilotinib treatment may represent a promising strategy to enhance elimination of residual LSC in TKI-treated CML patients. Disclosures:Buonamici:Novartis: Employment. Manley:Novartis: Employment. Holyoake:Novartis: Consultancy, Research Funding. Copland:Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bhatia:Novartis: Consultancy, Honoraria.

The Role of Mel18 Overlaps with BMI1 In the Self-Renewal of Human Hematopoietic Stem Cells.

AbstractAbstract 2619Complex mechanisms regulate the ability of hematopoietic stem cells (HSCs) to self-renew, some of which may be exploited by leukemic stem cells. BMI1, a member of the polycomb group (PcG) proteins is known to be a positive regulator of this process, largely by repressing the p16/INK4a locus. However the role of other PcG proteins is unclear. We initially screened HSCs and progenitor populations from umbilical cord blood (CB), for the expression patterns of a number of PcG genes. Levels of expression were heterogeneous, indicating that there may be different roles for different PcG in HSCs versus progenitors. As a starting point we have focused on Mel18 (PCGF2) as it has been suggested in murine hematopoiesis that it acts to counteract BMI1. Lineage negative CB cells were transduced with lentiviral vectors expressing shRNA against Mel18, BMI1 and a control sequence. Specific knockdown by these constructs was confirmed at the RNA level to be at least 80% for both genes and was verified at the protein level by Western blot. Our data indicates that knockdown of Mel18 impairs the proliferation of primitive cord blood cells in both stromal-dependent and -independent culture, in a similar manner to BMI1. Furthermore Mel18 deficiency impedes both primary and secondary colony formation of all myeloid lineages in methylcellulose. These findings have been confirmed in vivo with significant reduction in engraftment of CB lineage negative cells in NOD/SCID mice at twelve weeks. We are investigating whether over expression of Mel18 can rescue BMI1 deficient cells to establish if these homologous genes have redundant functions. The data so far suggests that BMI1 is not unique in its role in HSC self - renewal, and Mel18 may share overlapping functions. This highlights a possible difference between human and murine hematopoiesis. Moreover it is likely that other members of the PcG family are also important in human HSCs. It will be of interest to investigate whether like BMI1, they are also implicated in the maintenance of the leukemic stem cell. Disclosures:No relevant conflicts of interest to declare.