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Secondary acute myeloid leukemia (sAML) is the most therapeutically recalcitrant form of AML with a life expectancy of less than 12 months. Secondary AML evolves from relatively prevalent myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS), or after chemotherapy, radiation therapy, or hematopoietic cell transplantation (HCT) that together confer a 14% risk of sAML at 15 years. Cumulative sequencing studies show that human splicing factor mutations, epigenetic spliceosome deregulation, RNA editing-induced splicing alterations, and pro-survival splice isoform switching drive dormant leukemia stem cell (LSC) generation and sAML resistance to chemotherapy and molecularly targeted agents resulting in high rates of relapse. LSC are immunologically silent in part because they activate adenosine deaminase acting on dsRNA (ADAR1), which attenuates the innate immune response. In addition, therapeutic splicing modulation has the potential to induce neoepitope formation and augment checkpoint inhibitor therapy. Thus, there is a pressing need for clinical development of splicing modulatory agents that eradicate therapy resistant LSC and reduce sAML drug resistance and relapse. Rebecsinib (17 S-FD-895) is a pharmacologically stable, potent, and selective small molecule splicing modulator that targets the SF3B core of the spliceosome at the interface of SF3B1, SF3B3 and PHF5A. We previously showed that Rebecsinib inhibits human LSC maintenance in sAML models at doses that spare normal hematopoietic stem and progenitor cells (HSPCs). In IND-enabling studies, we now demonstrate that splicing modulation with this potent agent is a pre-clinical tox-proven strategy to eradicate LSC with the potential to overcome immune checkpoint resistance via inhibition of ADAR1 splicing and activity. We further describe targeted LSC eradication that correlates with detection of unique intron-retained and exon-skipped transcripts that can be quantified by splice isoform-specific qRT-PCR and RNA-sequencing analyses and can be used as predictive biomarkers to monitor molecular responses to Rebecsinib treatment. Mechanistically, the therapeutic effects were accompanied by on-target splicing modulatory effects, including reductions in pro-survival MCL1L transcripts and splicing factor gene products such as SF3B1 and SF3B3, which form part of the splicing modulator binding pocket as well as alterations in self-renewal promoting ADAR1 and STAT3beta transcripts. In multi-species toxicology and pharmacokinetic/pharmacodynamic studies, Rebecsinib induced splicing modulation and was well-tolerated over a broad range of doses. Because of disrupted spliceosome function, SF3B1 overexpression and increased dependence on pro-survival splice isoform expression, Rebecsinib-mediated induction of pro-survival to pro-apoptotic splice isoform switching inhibits sAML LSC survival and self-renewal at doses that spare normal HSPCs in vitro and in humanized mouse models commensurate with dose-dependent changes in splicing reporter exon skipping and SF3B1, MCL1, BCL2 and CD44 isoform levels. Together, this potent and selective agent along with biomarkers of response to splicing modulation provide a sensitive method of detecting activity and mechanism of action of Rebecsinib, and demonstrate its LSC selectivity in humanized stromal co-cultures and humanized mouse models, which will have utility in future clinical development of this novel therapeutic agent. Disclosures Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p < 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p < 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p < 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p < 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p < 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p < 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

SummaryLeukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

Summary Adenosine deaminase associated with RNA1 (ADAR1) deregulation contributes to therapeutic resistance in many malignancies. Here we show that ADAR1-induced hyper-editing in normal human hematopoietic progenitors impairs miR-26a maturation, which represses CDKN1A expression indirectly via EZH2, thereby accelerating cell-cycle transit. However, in blast crisis chronic myeloid leukemia progenitors, loss of EZH2 expression and increased CDKN1A oppose cell-cycle transit. Moreover, A-to-I editing of both the MDM2 regulatory microRNA and its binding site within the 3′ UTR region stabilizes MDM2 transcripts, thereby enhancing blast crisis progenitor propagation. These data reveal a dual mechanism governing malignant transformation of progenitors that is predicated on hyper-editing of cell-cycle-regulatory miRNAs and the 3′ UTR binding site of tumor suppressor miRNAs.

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.

SummaryThe oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Abstract 3739 Leukemia stem cells (LSC) represent a frequently dormant self-renewing population integral to the initiation, maintenance, and progression of human chromic myeloid leukemia (CML). The current standard of care dasatinib, a BCR-ABL targeted tyrosine kinase inhibitor (TKI), effectively eradicates the bulk of CML cells but frequently fails to affect the LSC population that is thought to drive CML relapse. Members in the BCL2 family are proteins that regulate apoptosis, 6 of which regulate cell survival. Each of these 6 members has a long and short isoform with opposing functions; generally, long isoforms promote cell survival while the short isoforms promote apoptosis. Previously, we demonstrated that upregulation of pro-survival BCL2 proteins in CML LSC contributes to chemotherapy resistance and LSC quiescence in protective hematopoietic niches. LSC found in different hematopoietic niches differ in their response to TKI treatment. Niche affects LSC cell cycle, either by maintaining quiescence or by promoting rapid cell cycling. Quiescent cells are a hurdle for traditional chemotherapy, which usually targets rapidly cycling cells, leaving the quiescent LSC untouched. We hypothesize that the inhibition of pro-survival BCL2 protein family members will sensitize LSC to dasatinib therapy and therefore prevent CML relapse. We tested a novel pan pro-survival BCL2 family protein inhibitor, sabutoclax, delivered by intravenous injection either alone or in combination with oral dasatinib in immunodeficient RAG2−/-gc−/- mice engrafted with BC CML patient samples. After treatment, LSC burden, self-renewal, and cell cycle status were quantified using FACS. Our results showed a reduction in the LSC burden in combination treated mice when compared to mice that received either drug alone. Mice treated with the combination regimen were found to have fewer quiescent human leukemic cells than their counterparts that received single agent treatments. Immunofluorescence staining confirmed the reduction of quiescent cells in the bone marrow after combination treatment when compared to single agent or vehicle treatments. We validated the molecular targets by using human specific splice isoform primers to perform RT-qPCR on FACS sorted LSC and showed a reduction in the BCL2 long to short isoform ratio in sabutoclax versus vehicle treated animals, indicating a skewing towards the pro-apoptotic splice variant. Together, these results indicate that the combination strategy with a pan pro-survival BCL2 family inhibitor and a tyrosine kinase inhibitor may be the foundation for a promising clinical strategy to effectively eliminate LSC and prevent cancer progression and relapse. Disclosures: No relevant conflicts of interest to declare.

Introduction Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are generated from progenitors that have aberrantly activated self-renewal pathways thereby resulting in tyrosine kinase inhibitor (TKI) resistance. The telomerase complex, consisting of a reverse transcriptase subunit (TERT), an RNA template subunit (TERC), and a protective shelterin scaffold, transcriptionally modulates the Wnt/b-catenin self-renewal pathway. Many malignancies, including BCR-ABL TKI resistant blast crisis CML (BC CML), exhibit robust telomerase activity thereby prompting the development of imetelstat, a competitive inhibitor of telomerase enzymatic activity. Imetelstat is a covalently lipidated 13-mer oligonucleotide that binds with high affinity to the TERC subunit. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms. However, the role of imetelstat in selective self-renewing LSC inhibition in CML had not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized LSC primagraft studies to investigate the capacity of imetelstat to selectively inhibit LSC self-renewal and to determine the mechanism of action. Methods and Results Cytoscape analysis of RNA-seq data derived from FACS-purified progenitors from human blast crisis (BC; n=9) compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples revealed transcriptional upregulation of b-catenin, LEF1, TCF7L1, ABL1 and other key genes within the TERT interactome suggesting a role for TERT activation in human BC LSC generation. Human progenitor LSC-supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 mM significantly inhibited (p Conclusions Niche responsive interactions between the telomerase complex and the Wnt/b-catenin self-renewal pathway sensitize b-catenin activated LSC to imetelstat in both in vitro and in vivo humanized pre-clinical BC CML models thereby providing a strong rationale for LSC eradication trials involving imetelstat. Disclosures Huang: Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Jamieson:UC San Diego: Other: I received funding from Janssen Research & Development, LLC.

Background Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge. Methods To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro. Results We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses. Conclusion Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.

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.

High-mobility group box 1 protein (HMGB1), a DNA-binding nuclear and cytosolic protein, is a pro-inflammatory cytokine released by monocytes and macrophages. HMGB1 as well as its B box domain induce maturation of human dendritic cells (DCs). This report demonstrates that the B box domain induces phenotypic maturation of murine bone marrow-derived dendritic cells (BM-DCs) as evidenced by increased CD86, CD40 and MHC-II expression. The B box domain enhanced secretion of pro-inflammatory cytokines and chemokines: IL-1beta, IL-2, IL-5, IL-8, IL-12 and tumor necrosis factor (TNF)-alpha, but not IL-6 and IL-10. Furthermore, four peptides whose sequences correspond to different regions of HMGB1 induced production of IL-1beta, IL-2 and IL-12 (p70), but not IL-10 and IL-6 in mouse BM-DCs. Interestingly, these peptides differed in their capacity to induce TNF-alpha, IL-5, IL-18 and IL-8. B box domain as well as peptide-activated DCs acted as potent stimulators of allogeneic T cells in a mixed leukocyte reaction. DCs exposed to HMGB1 peptides induced proliferation of ovalbumin-specific syngeneic T cells. These DC-activating peptides could serve as an adjuvant in immunotherapeutic or vaccine context and the selective activity of these different peptides suggests a means to customize the functional properties of DCs.

Abstract 3776 Cumulative evidence suggests that dormant self-renewing leukemia stem cells (LSC) contribute to relapse and blast crisis transformation by evading therapies that target cycling cells. Previously, sonic hedgehog (Shh) signaling was shown to modulate cell cycle regulation and self-renewal in normal mouse hematopoietic stem cells. However, its role in human LSC regeneration and quiescence had not been elucidated. Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−gc−/− mice with a selective Shh inhibitor, PF-04449913, reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation. Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory genes expression and splice isoform analysis demonstrated reversion towards a normal splice isoform signature for many cell cycle regulatory genes. Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected. Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators. Our findings suggest that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel LSC splice isoform detection platforms to assess efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapy may inform dormant cancer stem cell elimination strategies that ultimately avert relapse. Disclosures: Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership.

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.

Abstract 910 In blast crisis transformation of CML (BC CML), the leukemia stem cells (LSC), via the acquisition of both enhanced survival and self-renewal capacity, become increasingly resistant to BCR-ABL targeted tyrosine kinase inhibition and thus often contribute to relapse after treatment, pointing to the need for alternative therapeutic strategies and a better understanding of the molecular mechanisms underlying disease progression. Janus kinase 2 (JAK2) plays an important role in BCR–ABL + cell survival and has profound effects on self-renewal and lineage commitment of normal and leukemic hematopoietic stem cells, through the activation of the transcription factor signal transducer and activator of transcription 5 (STAT5). To determine if JAK/STAT signaling pathway activation is related to CML progression, LSC from human Chronic Phase (CP CML) and BC CML samples were sorted using FACS Aria (Lin-CD34+CD38+) and analyzed using splice-isoform specific q-RT-PCR. Our results showed that, compared to CP CML, BC LSC harbor enhanced mRNA expression of BCR-ABL, JAK2 and STAT5A isoforms, confirming that progression of CP to BC, in CML LSC, is marked by activation of JAK/STAT pathway. Therefore, we investigated the response of BC CML LSC to a clinical grade JAK2 inhibitor, SAR302503 (Sanofi, Cambridge, MA) alone or in combination with a potent BCR-ABL inhibitor, dasatinib, in vivo. After two weeks of treatment, RAG2−/−gc−/− mice intrahepatic transplanted with BC LSC, showed a significant (p To test whether the combination therapy can impair self-renewal capacity of the BC CML LSC in vivo, we immunomagnetic bead selected CD34+ cells from BM and spleens of treated mice, and serially transplanted an equal number into secondary recipients. We observed a significant (p Validation studies, using nanoproteomic analysis, confirmed that LSC sorted cells from mice treated with SAR302503 had lower expression levels of p-JAK2 (Tyr 1007-08) and p-STAT5A (Tyr 694) compared with vehicle treated mice (51% and 64% of reduction, respectively), while no changes are observed for total JAK2 protein or B2M between both conditions. Full transcriptome sequencing and q-RT-PCR analysis, on sorted CML LSC from mice treated with SAR302503 in combination with dasatinib, confirmed that STAT5A specific isoforms decresed after treatment, suggesting JAK/STAT pathway could be used as biomarker of response and could explain the impairment of self-renewal in the combination therapy. Disclosures: No relevant conflicts of interest to declare.

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.

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.

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.

Abstract 1507 Leukemia initiating cells (LIC) contribute to therapeutic resistance as a result of their capacity to accumulate mutations in pathways, such as the NOTCH1 receptor signaling pathway, that promote self-renewal and survival within specific niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in driving therapeutic resistance. However, the role of NOTCH1 activation in human T-ALL LIC propagation and LIC sensitivity to selective NOTCH1 receptor inhibition has not been examined. The difficulties in maintaining primary cultures of leukemia cells have hampered investigations into the biology of T-ALL LIC and underscore the need for a direct transplantation model to characterize human LIC in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL LIC. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2Rƒn gamma null (NSG) mice, was identified in primary adult T-ALL without an in vitro expansion. In this study, we identified and molecularly characterized potential LIC populations in pediatric T-ALL without preceding in vitro culture and examined the role of NOTCH1 activation in LIC propagation. To further define the T-ALL LIC, CD34+CD2+CD7+ or CD34+CD2+CD7− cells were isolated from T-ALL primary patients' blood by FACS sorting and transplanted into neonatal RAG2−/− gamma chain−/− mice to determine their leukemic engraftment potential. Limiting dilution experiments were performed with cells from six T-ALL patient samples. Mice transplanted with CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale bone marrow and enlarged spleen, thymus and liver. Hematopoietic organs were analyzed by flow cytometry and showed engraftment of bone marrow, spleen, thymus and liver. Furthermore, the disease could be serially transplanted. LIC were uniquely susceptible to targeted inhibition in vivo with a therapeutic human NOTCH1 negative regulatory region selective monoclonal antibody (mAb) while normal human hematopoietic progenitors were spared thereby highlighting the cell type and context specific effects of NOTCH signaling. Both the NOTCH1 mAb treatment and lentiviral shRNA knockdown of NOTCH1 reduced NOTCH1, HES1 and c-MYC transcript levels, underscoring the selectivity of NOTCH1 mAb inhibition of NOTCH signaling. These results demonstrate that CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL. Moreover, inhibition of NOTCH signaling by either mAb or shRNA-mediated Notch1 knockdown might be another strategy to target the LIC in T-ALL. Disclosures: No relevant conflicts of interest to declare.

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.

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.

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.