ALBERT

Description: Abstract 1848 Poster Board I-874 Multiple myeloma (MM) is the second most common hematologic malignancy and characterized by clonal proliferation of CD138+ bone marrow plasma cells. Despite various treatment options few patients with MM have been cured. Furthermore, high relapse rates and recent evidence from xenogeneic transplantation models and primary MM marrow samples indicate that a rare population of cells or MM cancer stem cells (MM CSCs) within the marrow regenerates itself and may be responsible for drug resistance. These MM CSCs are phenotypically similar to memory B cells (CD138- CD34-CD19+) but differ in that they have the capacity to regenerate themselves or self-renewal. However, most of the reports on MM CSC animal models are established in NOD/SCID mice that require a larger number (1 – 10 × 106) of bead sorted cells for each animal. In addition, the latency of MM induction (4 – 6 months) in NOD/SCID mouse models and lack of in vivo tracking of the malignant clone preclude robust pre-clinical testing of novel therapeutic strategies that target MM CSC. Mononuclear cells were isolated from autologous mobilized peripheral blood of at least four primary MM patients after Ficoll gradient centrifugation followed by immunomagnetic bead depletion of CD34+ and CD138+ cells and/or further sorted using a FACSAria. The CD138-CD34- population was transduced with lentiviral luciferase GFP (GLF) and transplanted (10,000 to 106 cells per mouse) intrahepatically into neonatal RAG2-/- gamma chain-/- (RAG2-/-gc-/-) mice. Engraftment was compared to mice transplanted with either CD34+ or CD138+ cells. Mice were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that a relatively rare CD138- CD27+ population, resembling memory B cells (∼1.2%), persists in MM autografts and can engraft immunocompromised mice more rapidly and effectively than the CD138+ (Lin+) population of mature plasma cells. This data supports the persistence of CSCs despite high dose chemotherapy further underscoring the need for CSC targeted therapy. Bioluminescence was detected in live mice transplanted with as little as 60,000 cells of CD138- CD34- population and as soon as 4 weeks after transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+ and CD138+ population in bone marrow, spleen and liver and bioluminescence was also detected in the secondary transplantation of cells from MMCSC primary engraftment demonstrating the self-renewal capacity of this rare CD138- CD27+ population. Our results suggest that by utilizing a lentiviral GFP-luciferase system in a highly immunocompromised mouse strain fewer cells will be required to monitor MM engraftment and perhaps hasten disease development. Further studies to confirm the expression of selected IgG genes from myeloma cells and to characterize the self-renewal capacity with genes involved in developmental signaling such as sonic hedgehog and wnt pathways are underway. Disclosures: Goff: Coronado Biosciences: Research Funding.

Description: Chronic myeloid leukemia (CML) represents an important paradigm for identifying the molecular events that promote malignant reprogramming of progenitors into therapeutically recalcitrant leukemia stem cells (LSC) during blast crisis (BC) transformation. To elucidate mechanisms of human BC LSC generation, whole transcriptome RNA sequencing (RNA Seq), lentiviral BCR-ABL and JAK2 transduction, quantitative RT-PCR (qRT-PCR) and serial xenotransplantation studies were performed. In human BC LSC, RNA seq revealed extensive upregulation of inflammation-responsive genes in conjunction with JAK/STAT signaling pathway activation and splice isoform specific qRT-PCR uncovered a predilection for selective STAT5a isoform expression. While lentiviral BCR-ABL1 expression in cord blood progenitors enhanced JAK2 activation and expression of specific STAT5a splice isoforms, lentiviral human JAK2 overexpression globally activated inflammation-response genes and expression of adenosine deaminase RNA associated (ADAR1), a primate specific RNA editase previously shown to activate self-renewal in response to inflammation. Notably, inhibition of BC LSC self-renewal with dasatinib, a BCR-ABL inhibitor, combined with a potent JAK2 inhibitor, SAR302503, was associated with reduced STAT5a isoform expression and phospho-STAT5 activation as well as ADAR1 expression and activity. These results highlight a novel JAK/STAT pathway driven niche-responsive mechanism of human BC LSC generation that can be targeted, at least in part, with a selective JAK2 inhibitor and may be utilized as an RNA editing-based biomarker of cancer stem cell generation and therapeutic resistance. Disclosures: Jamieson: Sanofi: Consultancy.

Description: Abstract 1927 Multiple myeloma (MM) is characterized by clonal proliferation of CD138+ plasma cells in the bone marrow (BM) and remains an incurable disease. Recent identification of a rare population of MM cancer stem cells (MM CSC) is phenotypically similar to memory B cells (CD138- CD34- CD19+) but differs in that they have the self-renewal capacity within the BM and may be responsible for drug resistance. Preclinical testing of novel therapeutic strategies that target MM CSC requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. We have previously reported results on establishing a MM animal model by transplanting MM CSC from autologous mobilized peripheral blood of primary MM patients, transduced with lentiviral luciferase GFP (GLF) and transplanted intrahepatically (IH) into neonatal RAG2/gc double knock-out (RG-KO). Here we evaluate engraftment efficiency in consideration of BM microenvironment by comparing CD45+ human cell engraftment in mice transplanted either IH to neonates or intrafemorally (IF) to gamma-irradiated young adult mice. MM CSC were selected from isolated PBMC after Ficoll gradient centrifugation of fresh BM biopsy from two primary MM patients or from a human MM cell line, H929, followed by immunomagnetic bead depletion of CD34+ and CD138+ cells. The cells were transplanted into RG-KO mice ranging from 53,000 to 10⋀6 cells per mouse either IH or IF. Mice transplanted with GLF-transduced MM CSC were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that bioluminescence signal levels were detected in mice transplanted IF with 53,000 MM CSC per mouse even before 3 weeks by ventral view and as early as 5 weeks by lateral view. To date, tumor growth was only discovered in mice transplanted IH with 2 × 10⋀6 unselected MM PBMC from a fresh BM biopsy as early as 10 week post-transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+, CD19+ and CD138+ population in tumor, bone marrow, spleen and liver. In addition, expression of clonal light chain restriction in myeloma cells confirmed myeloma engraftment. Future studies will focus on expression of genes involved in sonic hedgehog pathway as analyzed by PCR to confirm the self-renewal capacity. Moreover, investigations on the effect of B cell-activating factor (BAFF) in BM microenvironment by transplanting MM CSC into BAFFxRG-KO mice are in progress. Disclosures: Jamieson: Bristol-Meyers Squibb: Research Funding.

Description: Abstract 1470 To discover oncogenic pathways that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), we performed RNA interference screens both in T-ALL cell lines and primary specimens. We found that the JAK tyrosine kinase family member, TYK2, and its downstream effector, STAT1, are each required for the survival of T-ALL cells. To identify the effector molecules downstream of the TYK2-STAT1 pathway in T-ALL, we analyzed global gene expression profiles in TYK2-dependent T-ALL cell lines after silencing of TYK2 or STAT1. As expected, gene set enrichment analysis revealed that genes downregulated by TYK2 knockdown were generally also downregulated by knockdown of STAT1. Importantly, we found that expression of the anti-apoptotic gene BCL2 was significantly downregulated after silencing of both TYK2 and STAT1. Analysis by quantitative PCR of additional T-ALL cell lines revealed that silencing of TYK2 resulted in significant reductions of BCL2 mRNA expression in multiple TYK2-dependent cell lines. Expression of the wild-type but not the kinase-dead TYK2 protein was sufficient to rescue BCL2 protein expression and to prevent apoptosis after knockdown of endogenous TYK2, indicating that the tyrosine kinase activity of TYK2 is required for BCL2 upregulation. Similarly, expression of the shRNA-resistant wild-type STAT1A protein partially rescued BCL2 protein expression and prevented apoptosis, while a variant of STAT1A (Y701F) that is incapable of becoming phosphorylated on a requisite tyrosine residue did not rescue BCL2 levels. Taken together, our findings indicate that aberrant activation of a TYK2-STAT1 pathway upregulates BCL2 expression in T-ALL cells, and that the T-ALL cells develop pathway dependence, in that they require these sustained high levels BCL2 expression for survival. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1034 Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a therapeutically recalcitrant malignancy that accounts for approximately 15% of pediatric and 25% of adult ALL cases. In leukemia, cancer stem cells constitute a relatively rare population of tumor cells that play a key role in cancer propagation and, like adult stem cells, have enhanced self-renewal potential. A previous report showed that following in vitro culture, CD34+/CD4- and CD34+/CD7- subfractions of T-ALL marrow were enriched for leukemia stem cells (LSC) capable of engrafting leukemia in nonobese diabetic/severe combined immune deficient mouse (NOD/SCID). However, difficulties in maintaining primary cultures of leukemia cells hampered investigations into the biology of T-ALL underscoring the need for a direct transplantation model to characterize human LSC in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL. Experimental Procedures: Quantitative RT-PCR of NOTCH target gene expression and NOTCH mutation DNA sequencing analysis was performed on human CD34+ cells from T-ALL patient samples (n =12). To develop a humanized mouse model of T-ALL, CD34+ progenitors were lentivirally transduced with GFP-Luciferase Fusion protein (GLF) and transplanted intrahepatically into neonatal T, B, and NK cell deficient mice. In some experiments, FACS purified CD34+ subpopulations were transplanted at limiting dilution, including CD34+CD38+CD2+Lin- cells. Leukemic engraftment was monitored by in vivo bioluminescence imaging and analyzed by FACS detection of human CD34+ cells in liver, bone marrow, spleen and thymus when mice were sacrificed at 8–10 weeks post-transplant. NOTCH1 target gene expression was analyzed by q-RT-PCR in human CD34+ cells derived from engrafted tissues and NOTCH mutation analysis was performed by DNA sequencing on the same population. To assay LSC self-renewal, engrafted human CD34+ cells from bone marrow were transplanted into secondary and tertiary recipients. In serially transplanted mice, NOTCH1 target gene expression, NOTCH1 receptor expression was analyzed by FACS and NICD expression was assessed in the bone marrow by immunohistochemistry. Results: Q-RT-PCR data showed that NOTCH1, HES1 and c-MYC expression correlated with NOTCH 1 mutation status as well as the emergence of a CD34+CD2+Lin- population not evident in normal cord blood. We transplanted 12 T-ALL patient samples with detectable Notch1 expression and 100% of samples engrafted RAG 2-/- gamma c-/- mice. Transplanted LSC could be tracked for 10 weeks after transplant by in vivo bioluminescent imaging while Lin+ engraftment declined. Human CD34+/CD45+ cells, CD45+/CD34+/CD38+/Lin−/CD2+ cells were found in the bone marrow, thymus, spleen of the engrafted mice at 9–10 weeks post transplant or the end of dosing. Finally, human CD34+ cells engrafted secondary and tertiary recipients with T-ALL demonstrating their propensity for self-renewal and differentiation. Notch1 target gene and Hes1 expression was higher in patients with Notch1 mutation identified by sequencing. Conclusion: Serially transplantable candidate LSC retain high level NOTCH1 target gene expression and may be uniquely susceptible to targeted NOTCH1 receptor inhibition. Disclosures: Jamieson: Pfizer: Research Funding.

Description: Innate immune anti-viral adenosine to inosine (A-to-I) base editing enzymes (editases) promote hematopoietic stem cell (HSC) self-renewal and protect the human genome from retroviral integration in response to inflammatory cytokine signaling. However, hyper-editing has been linked to therapeutic resistance and cancer progression. Because myeloproliferative neoplasm (MPN) progression is typified by increased JAK2/STAT-mediated cytokine signaling, we investigated the cell type and context specific role of adenosine deaminase acting on RNA1 (ADAR1) editaseactivity in MPN pre-leukemia stem cell (pre-LSC) evolution into acute myeloid leukemia stem cells (LSCs). Here we show by whole transcriptome sequencing (RNA-seq) of 113 FACS-purified hematopoietic stem cells and progenitors from 78 individuals, including 54 MPN and AML patients and 24healthy young and aged individuals, that anti-viral signaling pathway activation and splice isoform switching from ADAR1p110 to JAK2/STAT-inducible ADAR1p150 RNA editase activation contributes to MPN progression. Pre-LSC evolution to LSC was characterized by ADAR1p150 upregulation, distinctive RNA editome patterns, STAT3 hyper-editing, increased replating as a measure of self-renewal. Moreover, LSC generation was typified by beta-catenin self-renewal pathway upregulation, which was recapitulated by lentiviral ADAR1p150 overexpression and reversed by lentiviral ADAR1p150 shRNA knockdown. Our studyunderscores the importance of inflammatory-cytokine fueled enzymatic mutagenesis in human MPN pre-LSC evolution to LSC. Thus, this study sets the stage for developing predictive RNA editome biomarkers of LSC generation to guidetherapeutic strategies aimed at preventing progression of hematopoietic malignancies. Disclosures Crews: Ionis Pharmaceuticals: Research Funding.

Description: To further unravel the molecular pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), we performed high-resolution array comparative genomic hybridization on diagnostic specimens from 47 children with T-ALL and identified monoallelic or biallelic LEF1 microdeletions in 11% (5 of 47) of these primary samples. An additional 7% (3 of 44) of the cases harbored nonsynonymous sequence alterations of LEF1, 2 of which produced premature stop codons. Gene expression microarrays showed increased expression of MYC and MYC targets in cases with LEF1 inactivation, as well as differentiation arrest at an early cortical stage of thymocyte development characterized by expression of CD1B, CD1E, and CD8, with absent CD34 expression. LEF1 inactivation was associated with a younger age at the time of T-ALL diagnosis, as well as activating NOTCH1 mutations, biallelic INK4a/ARF deletions, and PTEN loss-of-function mutations or activating mutations of PI3K or AKT genes. These cases generally lacked overexpression of the TAL1, HOX11, HOX11L2, or the HOXA cluster genes, which have been used to define separate molecular pathways leading to T-ALL. Our findings suggest that LEF1 inactivation is an important step in the molecular pathogenesis of T-ALL in a subset of young children.

Description: Abstract 1693 The aim of this study is to develop clinical strategies that will HALT progression of CML by reducing leukemia stem cell (LSC) burden using a clinical grade JAK2 inhibitor, SAR302503 (SAR503, Sanofi, Cambridge, MA), alone or in combination with a potent BCR-ABL inhibitor, dasatinib. For this, CML patient samples in blast crisis phase (BC CML) were subjected to immunomagnetic bead CD34 selection or FACS Aria ll sorted to obtain leukemic progenitors (LSC/CD34+CD38+Lin−). Malignant progenitors were then transplanted into neonatal RAG2−/−gc−/− mice, and 8 weeks post-transplant, mice were treated with SAR503, dasatinib and vehicle for 14 days. Following treatment, hematopoietic tissues were analyzed for human engraftment by FACS analysis. Our results revealed that single agent experiments with SAR503 had a cytostatic rather than a cytoreductive effect on BC LSC. The treatment alone (60 mg/kg twice daily administered by oral gavage) did not significantly reduce leukemic progenitor burden in the liver, spleen, bone marrow and peripheral blood. Conversely, combination therapy with SAR503 and dasatinib (50mg/kg/day) significantly reduced LSC progenitors in all tissues examined. Interestingly, we observed that dasatinib alone therapy reduced the LSC burden in the liver, spleen, and peripheral blood, but the bone marrow retained a significant population of BC LSC. Also we found that the GMP population, previously shown to be enriched for BC LSC (Jamieson et al NEJM 2004; Abrahamsson et al PNAS 2009), was preferentially localized in the bone marrow. As shown by our laboratory and others, LSC therapeutic resistance may be influenced by extrinsic cues provided by the niche (e.g. promoting quiescence). Because quiescence has been implicated in driving tyrosine kinase inhibitor resistance and LSC survival and because the bone marrow retains a resistant population, we decide to perform secondary transplantation experiments to determine relapse potential (self-renewal). LSC progenitors were isolated by immunomagnetic bead selection of human CD34+ cells from marrows and spleens of treated mice. After serially transplanting an equal number of this cells into secondary recipients, we observed a significant reduction in LSC serial transplantation only following combination treatment, suggesting that the combination therapy can abolish LSC self-renewal capacity and thereby potentially prevent relapse. To validate drug exposure, we have been performing both genomic and nanoproteomic analysis. Regarding the proteomics validation studies, we analyzed sorted LSC derived from spleen (pooled 5 mice per group) that were treated with vehicle or SAR503 for 14 days. The analysis was performed to detect status of p-JAK2, JAK2, p-STAT5 and B2-microglobulin (loading control). We observed a down regulation on the levels of p-JAk2 (active site Tyr 1007–08) and p-Stat5 (active site Tyr 694) (35% and 42% respectively), while no changes are observed for total JAK2 protein or B2M between both conditions. The full transcriptome sequencing, on sorted LSC treated with SAR503 alone and in combination with dasatinib, identified specific isoform changes in the JAK/STAT pathway that could be used as biomarkers of response and could explain the synergistic effect of the combination therapy. We have also characterized, at an isoform level, biomarkers of resistance that could explain relapse of disease after single agent therapy and we are currently validating these findings. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2735 Leukemia stem cells (LSC) play a crucial role in the development and progression of chronic myeloid leukemia (CML). Although BCR-ABL targeted tyrosine kinase inhibitors (TKI), such as dasatinib, can eradicate the majority of CML cells, they frequently fail to eliminate the dormant, niche-resident LSC that are hypothesized to drive CML relapse. Cumulative evidence from CML cell lines and CD34+ primary patient cells suggests that increased expression of pro-survival BCL2 family members contributes to TKI resistance and CML progression. However there is a relative dearth of data on BCL2 family expression in primary CML LSC and on the role of these proteins in TKI resistance in selective niches. Full transcriptome RNA sequencing revealed that LSC switch from pro-apoptotic to pro-survival BCL2 family member splice isoform expression during progression from chronic phase to blast crisis CML. Using splice isoform-specific qRT-PCR, we identified overrepresentation of long (pro-survival) compared with short (pro-apoptotic) MCL1, BCLX, and BCL2 isoforms in blast crisis LSC compared with chronic phase and normal progenitors. Following intrahepatic transplantation of blast crisis LSC into neonatal RAG2−/−gc−/− mice, LSC engrafted in the marrow niche were quiescent, were dasatinib resistant and upregulated BCL2 expression. These data led us to speculate that inhibition of BCL2 in dasatinib-resistant LSC may sensitize LSC to TKI therapy. Treatment with a high-potency, novel pan-BCL2 family inhibitor, sabutoclax, in vitro led to a dose-dependent increase in apoptosis along with a decrease in the frequency of leukemic progenitors compared to vehicle treated controls. Normal human cord blood progenitor cells were less sensitive to sabutoclax treatment with IC50 approximately five times higher than that for blast crisis CML cells (210 nM versus 43 nM). Moreover, sabutoclax treatment did not inhibit cord blood colony formation or colony replating in vitro. Treatment of CML LSC-transplanted mice with sabutoclax led to a significant reduction in LSC burden in all hematopoietic organs analyzed. Sabutoclax treatment in vivo also sensitized surviving bone marrow blast crisis LSC to dasatinib treatment ex vivo. Importantly, there was no reduction in normal progenitor engraftment in bone marrow following sabutoclax treatment. These results demonstrate that marrow niche blast crisis CML LSC survival is driven by overexpression of multiple pro-survival BCL2 family isoforms rendering them susceptible to a novel pan, BCL2 antagonist, sabutoclax, at doses that spare normal hematopoietic progenitors. While BCL2 splice isform switching promotes LSC survival and TKI resistance, pan-BCL2 family member inhibition with sabutoclax eliminates LSC and may form the cornerstone of a clinical strategy to avert cancer progression and relapse. Disclosures: No relevant conflicts of interest to declare.