Human B-ALL Research Articles

A Phase 1b, Open-Label, Dose Escalation and Expansion Study Evaluating the Safety and Efficacy of Entospletinib (GS-9973) with Vincristine and Dexamethasone in Adult Subjects with Relapsed or Refractory Acute Lymphoid Leukemia (ALL)

Background: Despite progress in understanding the biology and improvements in the treatment of ALL resulting in the majority of adults entering a complete remission (CR), ultimately most patients relapse. Allogeneic hematopoietic stem cell transplantation (HSCT) after initial cytoreduction in a second or subsequent CR offers a chance of cure in a small, highly selected group of subjects. Therefore, re-induction of a CR is the first step in the treatment of relapsed disease. Current treatments for relapsed ALL are generally unsuccessful and most die of their disease, underscoring the need for novel therapeutic approaches.Spleen tyrosine kinase (SYK) is a mediator of B-cell receptor signaling in normal and transformed B-cells. Increased activity of SYK in ALL cells may play a role in leukemia cell survival and proliferation. Deregulated SYK activity allows growth factor-independent proliferation and transformation of bone marrow-derived pre-B cells in mice. B-ALL results from the abnormal accumulation of transformed progenitor or precursor B lymphocytes (Wossning T, et al. JEM 2006), and inhibition of SYK has been shown to attenuate the growth of B-ALL in vitro and in vivo in mouse models. SYK activation occurs in human B-ALL and is sensitive to small molecule SYK inhibitors; in vitro treatment of primary patient-derived B-ALL cells with a SYK-inhibitor inhibits the proliferation of B-ALL cells (Perova T, et al. Sci Transl Med 2014).Entospletinib is an orally bioavailable, selective inhibitor of SYK which has shown tolerable safety and clinical activity in mature B-cell malignancies (Sharman J, et al. Blood. 2015). In pre-clinical models, the anti-leukemia effect of entospletinib was synergistic with vincristine (M. Axelrod, et al. ASH abstract 2015). We are conducting a Phase 1b trial (NCT02404220) evaluating entospletinib in combination with standard chemotherapy (vincristine and dexamethasone) for the treatment of relapsed or refractory adult Pre B-ALL (figure 1).Methods: This dose escalation trial will follow a 3+3 design. A subsequent dose expansion will enroll 15 additional subjects at the recommended dose level from the dose escalation.Key inclusion criteria for the trial include: previously treated B-ALL (Ph+ or Ph-), ≥18 years of age, ECOG PS≤2 with adequate organ function and resolution of all toxic effects of any prior therapy to ≤Grade 1.Key exclusion criteria include: diagnosis of mature B-ALL (Burkitts leukemia), active or symptomatic CNS disease, history of myelodysplastic syndrome or solid organ transplantation, allogeneic bone marrow progenitor cell transplant within 100 days or on active immunosuppression for GVHD, ongoing/active pneumonitis, and current therapy with proton pump inhibitors.Results: As of 7/21/2015, 3 patients have been enrolled into dose level 1, and 1 patient (64 y.o. female in 1st relapse) has completed the protocol-specified 2 induction cycles of treatment without dose-limiting toxicity. At the end of the 2nd induction cycle, that patient became transfusion independent and bone marrow evaluation showed morphologic CR (MRD positive) with recovery of normal hematopoiesis.Conclusions: The safety of entospletinib administered in combination with vincristine and dexamethasone is being investigated for the treatment of relapsed/refractory adult ALL. [Display omitted] DisclosuresStock:Gilead: Membership on an entity's Board of Directors or advisory committees. Crosswell:KIYATEC (employment); Gilead (common stock ownership and research funding): Employment, Equity Ownership, Research Funding. Chun:Gilead Sciences: Employment. Abella:Gilead: Employment. He:Gilead Sciences: Employment. Eng:Gilead: Employment. Douer:Gilead: Consultancy.

A Novel DLL1-Anti-CD19 Chimeric Protein Effectively Targets Notch Activation to B-Cell Acute Lymphoblastic Leukemia and Induces Cell Death

Background: Acute lymphoblastic leukemia (ALL) is the most prevalent form of cancer in children, and those who relapse continue to have poor survival. Therefore, the development of improved and specifically targeted treatment options is vital. The Notch pathway has been shown to act as a tumor suppressor in B-ALL via cell type specific induction of growth-inhibiting and pro-apoptotic pathways. In this study, we aim to therapeutically activate Notch signaling in B-ALL via targeting the Notch ligand DLL1 to B cells using an anti-CD19 scFv chimeric protein.Methods: A soluble chimeric protein composed of the extracellular domain of the Notch ligand DLL1 linked to a validated anti-CD19 scFv was isolated from HEK-293 producer cells. Human B-ALL (SB, Nalm6) and T-ALL (Jurkat) lines were treated with DLL1-anti-CD19scFv chimeric protein, and expression of the Notch target gene HES1 and effects on cell growth and survival were measured.Results: Both B- and T-ALL lines express Notch1 and Notch2 receptors on the cell surface. Exposure of B-ALL cells to DLL1-anti-CD19scFv chimeric protein led to an increase of Notch signaling, measured via 3-14 fold increase in HES1 mRNA expression. As expected, surface expression of the Notch receptors decreased upon chimera exposure, as Notch receptors are cleaved and destroyed upon activation. Importantly, exposure of the B-ALL lines to the chimeric protein led to a maximum 60% decrease in cell counts over 3-4 days, in contrast to T-ALL, where exposure did not effect growth significantly.Conclusions: This study demonstrates that Notch signaling can be feasibly activated in human B-ALL cells through a soluble DLL1-anti-CD19 scFv chimeric protein. Activation of Notch signaling via this method leads to growth inhibition and cell death in B-ALL, but not T-ALL cells. Based on our findings, we suggest that this soluble DLL1-anti-CD19 chimera may be a potential therapeutic approach for B-ALL, and further in vivo testing is warranted. DisclosuresNo relevant conflicts of interest to declare.

HIF1-Alpha and MYC Transcription Factor Signatures in B-Cell Acute Lymphoblastic Leukemia Are Associated with Positive Minimal Residual Disease Status: Therapeutic Implications

Precursor B-cell lymphoblastic leukemia (B-ALL) in adults remains a challenging clinical problem due to higher relapse rate and worse prognosis than in children. Although most of adult patients respond to standard induction therapy and complete remissions (CR) are typically achieved in 90% of patients, the majority of them eventually relapse. Our previous studies indicate that the minimal residual disease (MRD) status after induction therapy is the most important risk factor of relapse in adult B-ALL patients [Br J Haematol 2008;142:227-37]. We hypothesized that the survival of B-ALL blasts after induction therapy is a result of intrinsic characteristics of the tumor cells that determine resistance to chemotherapeutics. Therefore, we sought to identify the molecular background of B-ALL cells resistance to daunorubicin.To identify potential mechanisms responsible for drug-resistant phenotype, we utilized gene expression data from previous studies that assessed transcriptional profiles of drug-sensitive and drug-resistant cells. Using gene set enrichment analysis (GSEA) of daunorubicin-sensitive versus -resistant phenotypes of B-ALL cells we identified differential expression HIF1α and MYC transcription factors target genes (p=0.002, FDR=0.144; p<0.001; FDR=0.171, respectively). To verify these in silico findings, we compared the expression of a panel of MYC and HIF1α target genes in 41 newly diagnosed adult B-ALL patients who subsequently underwent standard induction therapy according to Polish Adult Leukemia Group PALG-ALL6 protocol. Expression of MYC and HIF1α signature genes was significantly higher in patients with positive (>0.1%) MRD status after completion of the induction therapy. Among studied HIF1α and MYC targets, lactate dehydrogenase A (LDHA) expression was the best predictor differentiating MRD+ versus MRD- patients (p=0.0019, FDR=0.005). It was of particular interest, since tumor stem cells are typically characterized by MYC and HIF1α transcriptional signatures, which rewire cellular metabolism towards aerobic glycolysis. We next assessed the effect of LDHA inhibition with a small molecule inhibitor, GSK2837808A, on proliferation and clonogenicity of human B-ALL cell lines. GSK2837808A markedly reduced lactate production in B-ALL cell lines (RS4;11, SEM-K2 and NALM-6) and decreased proliferation and colony formation in semi-solid medium in a dose-dependent fashion.Taken together, we show that adult B-ALL patients with positive MRD status after induction therapy exhibit concordant upregulation of HIF1α and MYC signature genes. Expression of LDHA, a target gene regulated by both HIF1α and MYC transcription factors was significantly higher in MRD-positive patients. Finally, inhibition of LDHA markedly decreased proliferation and clonogenicity of B-ALL cell lines, indicating that LDHA might be a therapeutic target in B-ALL. DisclosuresNo relevant conflicts of interest to declare.

BALR-6 regulates cell growth and cell survival in B-lymphoblastic leukemia.

BackgroundA new class of non-coding RNAs, known as long non-coding RNAs (lncRNAs), has been recently described. These lncRNAs are implicated to play pivotal roles in various molecular processes, including development and oncogenesis. Gene expression profiling of human B-ALL samples showed differential lncRNA expression in samples with particular cytogenetic abnormalities. One of the most promising lncRNAs identified, designated B-ALL associated long RNA-6 (BALR-6), had the highest expression in patient samples carrying the MLL rearrangement, and is the focus of this study.ResultsHere, we performed a series of experiments to define the function of BALR-6, including several novel splice forms that we identified. Functionally, siRNA-mediated knockdown of BALR-6 in human B-ALL cell lines caused reduced cell proliferation and increased cell death. Conversely, overexpression of BALR-6 isoforms in both human and mouse cell lines caused increased proliferation and decreased apoptosis. Overexpression of BALR-6 in murine bone marrow transplantation experiments caused a significant increase in early hematopoietic progenitor populations, suggesting that its dysregulation may cause developmental changes. Notably, the knockdown of BALR-6 resulted in global dysregulation of gene expression. The gene set was enriched for leukemia-associated genes, as well as for the transcriptome regulated by Specificity Protein 1 (SP1). We confirmed changes in the expression of SP1, as well as its known interactor and downstream target CREB1. Luciferase reporter assays demonstrated an enhancement of SP1-mediated transcription in the presence of BALR-6. These data provide a putative mechanism for regulation by BALR-6 in B-ALL.ConclusionsOur findings support a role for the novel lncRNA BALR-6 in promoting cell survival in B-ALL. Furthermore, this lncRNA influences gene expression in B-ALL in a manner consistent with a function in transcriptional regulation. Specifically, our findings suggest that BALR-6 expression regulates the transcriptome downstream of SP1, and that this may underlie the function of BALR-6 in B-ALL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0485-z) contains supplementary material, which is available to authorized users.

A minimum attention control law for ball catching

Digital implementations of control laws typically involve discretization with respect to both time and space, and a control law that can achieve a task at coarser levels of discretization can be said to require less control attention, and also reduced implementation costs. One means of quantitatively capturing the attention of a control law is to measure the rate of change of the control with respect to changes in state and time. In this paper we present an attention-minimizing control law for ball catching and other target tracking tasks based on Brockett’s attention criterion. We first highlight the connections between this attention criterion and some well-known principles from human motor control. Under the assumption that the optimal control law is the sum of a linear time-varying feedback term and a time-varying feedforward term, we derive an LQR-based minimum attention tracking control law that is stable, and obtained efficiently via a finite-dimensional optimization over the symmetric positive-definite matrices. Taking ball catching as our primary task, we perform numerical experiments comparing the performance of the various control strategies examined in the paper. Consistent with prevailing theories about human ball catching, our results exhibit several familiar features, e.g., the transition from open-loop to closed-loop control during the catching movement, and improved robustness to spatiotemporal discretization. The presented control laws are applicable to more general tracking problems that are subject to limited communication resources.

Abstract 4202: Targeting focal adhesion kinase is a novel approach to therapy of high-risk, Ikaros-mutant acute B-cell lymphoblastic leukemia

Abstract Deletions and dominant-negative mutations in IKZF1, the gene encoding Ikaros transcription factor, are found in ∼85% of Ph+ B-ALL and in some cases of Ph− B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph− or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and ponatinib have been incorporated into chemotherapy regimens for Ph+ B-ALL, the majority of patients still relapse, which correlates with residual bone marrow disease following induction therapy. New therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph− B-ALL. Using mice with a conditional Ikzf1 mutation (Ike5fl) that mimics the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated that loss of Ikaros DNA-binding function arrests B-lymphoid development at a large pre-B cell stage that can give rise to B-ALL. Survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK downstream of integrins and growth factor receptors that plays important roles in cancer stem cell biology and the tumor microenvironment. Here, we show that dysregulated tyrosine kinases, including BCR-ABL1, cooperate with Ikaros mutation to accelerate the development of B-lymphoblastic leukemia in mice, correlated with a striking (∼30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Ikzf1-mutant BCR-ABL1+ lymphoblasts exhibit relative resistance to ABL1 TKIs including dasatinib that is dependent on the presence of stroma. VS-4718 is a potent, orally bioavailable FAK inhibitor that inhibits tumor growth and metastasis in preclinical models, and is currently under evaluation in clinical trials in patients with various solid tumors. VS-4718 treatment abolished stromal adhesion and induced apoptosis of Ikzf1-mutant B-ALL and synergized with dasatinib, but had little effect on Ikzf1 WT B-ALL cells. Primary human Ph+ B-ALL samples showed a correlation between IKZF1 mutation status, stromal adherence, and sensitivity to FAK and ABL inhibitors in vitro, and BM tropism in vivo in xenografted NSG mice. Results from ongoing in vivo therapy experiments will be presented. Together, these results suggest a new model for the pathogenesis of high-risk B-ALL and its resistance to conventional therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM leukemic stem cells. Inhibition of FAK signaling in Ph+ or Ph− IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. FAK inhibitors represent a promising new approach for the treatment of high-risk IKZF1-mutant B-ALL patients. Citation Format: Nilamani Jena, Ila Joshi, Toshimi Yoshida, Zhihong Zhang, Zhong-Ying Liu, Prasanthi Tata, Irina M. Shapiro, Jonathan A. Pachter, David T. Weaver, Katia Georgopoulos, Richard A. Van Etten. Targeting focal adhesion kinase is a novel approach to therapy of high-risk, Ikaros-mutant acute B-cell lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4202. doi:10.1158/1538-7445.AM2015-4202

27. Aberrant Expression of the Stem Cell microRNA-126 Induces B Cell Malignancy

MicroRNAs are essential regulators of normal and malignant hematopoiesis. miRNAs are relevant for gene therapy, since they can be exploited to fine-tune the expression profile of vector constructs or to alter viral tropism (GentnerN Chiriaco et al, 2014; Escobar et al, 2014) and described the function of miR-126 in HSC where it regulates the balance between quiescence and self-renewal (Lechman et al, 2012). We here report a novel role for miR-126 in the induction and maintenance of high-grade B cell malignancies. By ectopically expressing miR-126 in transplanted BM cells, we observed that up to 60% of mice (n=71) developed B cell malignancies. LV insertion site (IS) analysis revealed that all tumors were monoclonal. We then tracked back leukemic clone to different hematopoietic lineages prospectively purified from the mice 2-6 months before disease onset. IS sharing between normal lineages and leukemic clone suggests stem or multipotent progenitor cell as origin for most tumors. Importantly, we show that miR-126 is the direct cause of genesis and maintenance of leukemia, since leukemogenesis is abolished when miRNA expression is inhibited by doxycycline (doxy) using a tetracycline-repressible miR-126 cassette, and established symptomatic leukemia completely regresses when miR-126 is switched off by doxy through induction of apoptosis. Transcriptional profiling indicated that miR-126 regulates multiple genes in p53 pathway both in murine blasts and in normal human CD34+ cells. Previous work suggested expression of miR-126 in acute lymphoblastic leukemia (ALL) and germinal center lymphoma. To further establish the relevance of miR-126 in human disease, we measured miR-126 expression in blasts from 16 adult patients with ALL. miR-126 was highly expressed in most studied ALL cases (Phil+: n=11, Phil-: n=5), at similar levels as CD34+ cells. We then down-regulated miR-126 in primary blasts from human B-ALL patients (n=5), and we observed increased apoptosis and impaired engraftment in xenograft models after primary and secondary transplantation (miR-126/KD: n=32 mice; Ctrl: n=37 mice), demonstrating the relevance of miR-126 in human B-ALL. In conclusion, we present a novel spontaneous mouse model for high grade B cell malignancies which are addicted to miR-126 expression, provide insight into the dynamic process of leukemogenesis by clonal IS tracking and unveil key tumor signaling pathways controlled by miR-126. Down-regulation of miR-126 could be exploited as therapeutic strategy in ALL, since it would deplete leukemic cells while expanding normal HSC, two ways to restore normal hematopoieis.

Type II JAK2 Inhibitor NVP-CHZ868 Is Active in Vivo Against JAK2-Dependent B-Cell Acute Lymphoblastic Leukemias (B-ALLs)

Approximately 10% of B-ALLs harbor CRLF2 rearrangements, which may portend a poor prognosis. Although these leukemias are addicted to JAK2 signaling, ATP-competitive type I JAK2 inhibitors have limited activity in vitro or in vivo (Weigert et al. J Exp Med 2012). This may result from heterodimerization of JAK2 with other JAK family members (Koppikar et al. Nature 2012). Type II inhibitors bind JAK2 in the inactive conformation, which may overcome this resistance. When assayed in MHH-CALL4 cells harboring a CRLF2/IGH rearrangement and JAK2 I682F mutation, the type II JAK2 inhibitors NVP-BBT594 and NVP-CHZ868 were 10-35-fold more potent than the type I JAK2 inhibitors NVP-BSK805 and NVP-BVB808. Similarly, in Ba/F3 cells dependent on CRLF2 and the gain-of-function allele JAK2 R683G, the IC50 for CHZ868 was 5-20-fold lower than the IC50s for BSK805 and BVB808. Unlike type I inhibitors, which induce paradoxical hyperphosphorylation of JAK2, CHZ868 completely blocks JAK2 and STAT5 phosphorylation. In addition, the JAK2 Y931C allele that confers 4-6-fold resistance to BSK805 and BVB808 did not alter sensitivity to CHZ868. CHZ868 abrogates STAT5 phosphorylation in Ba/F3 cells expressing CRLF2 with JAK2 R683G/Y931C while BVB808 does not. CHZ868 is the first type II JAK2 inhibitor amenable to in vivo use. We assessed its efficacy in mice transplanted with transgenic (CRLF2/JAK2 R683G/Cdkn2a-/- or CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr) or primary human CRLF2-rearranged B-ALLs. Splenocytes from patient-derived xenografts (PDXs) treated with CHZ868 in vivo for 3 days are more primed for apoptosis as demonstrated by a 2-6-fold EC50 reduction for PUMA permeabilizing activity compared to vehicle. Transcriptional profiling of splenocytes from CHZ868-treated PDXs revealed downregulation of critical survival pathways including E2F1, STAT3, and AKT-mediated signaling. Of note, 2 of the most downregulated genes are STAT targets, PIM1 and Myc. Mice treated for 5-6 days with CHZ868 had significant reductions in spleen size and complete loss of phospho-STAT5 in residual leukemia cells. In both murine leukemias and human xenografts, CHZ868 prolonged survival compared to controls (p<0.001). BH3 profiling of splenocytes from PDXs treated until moribund showed a 2-4-fold increase in the EC50 for BIM compared to vehicle, consistent with decreased priming for apoptosis in the relapsed setting. To study mechanisms of resistance to type II JAK2 inhibitors, we screened a randomly mutagenized JAK2 R683G library expressed in Ba/F3-CRLF2 cells for clones resistant to BBT594. All >30 clones sequenced harbored the same JAK2 L884P mutation. Ba/F3 cells expressing CRLF2 with JAK2 R683G/L884P displayed cross-resistance to CHZ868, while sensitivity to type I inhibitors was not affected. Structural modeling of the JAK2 JH1 domain suggested that L884P alters the binding pocket for type II inhibitors. JAK2 L884P is homologous to an EGFR L747P activating mutation, which destabilizes the P-loop and C-helix portion of the kinase domain (He et al. Clin Cancer Res 2012). The fact that L884P was reported in two B-ALL patients lacking additional JAK2 mutations (Torra et al. Blood (ASH Annual Meeting Abstracts) 2010) raised the possibility it was also an activating mutation. We confirmed L884P is an activating allele, as Ba/F3 cells expressing CRLF2, IL7R, and JAK2 L884P proliferated in the absence of TSLP ligand. To improve CHZ868 efficacy, we tested for synergy with multiple chemotherapy agents currently used in B-ALL treatment. Dexamethasone was the most highly synergistic with CHZ868 in MHH-CALL4 cells. To assess the combination in vivo, we treated mice transplanted with CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr murine B-ALL with vehicle, CHZ868, dexamethasone, or CHZ868 + dexamethasone for 14 days post engraftment. CHZ868 treatment prolonged survival compared to vehicle (p<0.0001) or dexamethasone (p<0.01), and the combination prolonged survival beyond CHZ868 monotherapy (p<0.0001). In summary, the type II JAK2 inhibitor CHZ868 potently kills JAK2-dependent B-ALL and overcomes genetic resistance to type I inhibitors. CHZ868 prolongs survival in murine transgenic and human xenograft models and synergizes with dexamethasone in vivo. Thus, combination strategies using dexamethasone with type II JAK2 inhibitors merit testing in patients with relapsed or refractory JAK2-dependent B-ALL. DisclosuresHofmann:Novartis Institutes for BioMedical Research: Employment. Baffert:Novartis: Employment. Vangrevelinghe:Novartis Institutes for BioMedical Research: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Weinstock:Novartis: Consultancy, Research Funding.

G-CSF Augments Inotuzamab-Mediated Clearance of Acute Lymphoblastic Leukemia from the Bone Marrow

Preclinical studies previously reported by our group at ASH suggest that treatment with granulocyte-stimulating factor (G-CSF) may provide a potent and well-tolerated method to disrupt the ‘leukemia cell niche’. Specifically, our data show that G-CSF treatment in mice reprograms bone marrow stromal cells, markedly inhibiting their expression of key chemokines/cytokines that support B lymphopoiesis, including stromal derived factor-1 (SDF-1/ CXCL12), interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), and interleukin-7 (IL-7). These changes in the bone marrow microenvironment result in a more than 10-fold decrease in B cells in the bone marrow, including early B cell precursors. Preliminary studies in humans suggest that G-CSF has a similar effect on the bone marrow microenvironment in healthy subjects. Since these B trophic factors also have been implicated in the maintenance of B cell acute lymphoblastic leukemia (ALL) cells, we hypothesize that G-CSF treatment in patients with acute lymphoblastic leukemia (ALL) will generate a ‘hostile’ bone marrow microenvironment for ALL cells, depriving them of key support signals and rendering them more sensitive to cytotoxic therapy.Herein, we specifically test whether niche disruption with G-CSF augments inotuzumab-mediated ALL cell clearance. Inotuzumab ozogamicin (Pfizer) is a CD22-calicheamicin antibody-drug conjugate. Initial clinical trials have documented activity of inotuzumab in B-ALL, with an overall response rate of 57% in relapsed or refractory B-ALL. To test this hypothesis, we developed a xenotransplantation model of human ALL using the human B-ALL G2 cell line, which expresses moderate levels of CD22. G2 cells transduced with a GFP-click beetle red luciferase retroviral construct to allow for in vivo bioluminescent imaging were injected intravenously into NOD/SCID/γc-/- (NSG) mice. The G2 cells preferentially home to the bone marrow but eventually metastasize to other organs, including the brain. Four cohorts of mice were studied (n = 9-21, each), including: 1) G2 ALL cells alone; 2) inotuzumab alone (single injection of 80 µg/kg, IP); 3) G-CSF alone (100 µg/kg/day for 14 days by continuous subcutaneous infusion); or 4) G-CSF plus inotuzumab (G-CSF was started four days prior to inotuzumab). Treatment with inotuzumab increased median survival from 25.5 days to 41 days (P < 0.0001). G-CSF alone had no effect on survival. However, combination of G-CSF plus inotuzumab further increased survival to 49 days (P = 0.01 compared with inotuzumab alone). G-CSF also improved survival in mice given two doses of inotuzumab (each 80 µg/kg, IP) 10 days apart. Bioluminescent imaging revealed that, in several of the mice in the G-CSF plus inotuzumab group, the bioluminescent signal was predominantly localized to the brain, with little or no signal in bone.These data are consistent with the possibility that inotuzumab may have limited penetration into the brain. Thus, the positive effect of combination G-CSF plus inotuzumab on survival may be limited by CNS disease in this model. Indeed, the addition of G-CSF to inotuzumab decreased the bioluminescent signal in bone regions on day 21 after inotuzumab by approximately 10-fold (Figure 1, P < 0.001). Collectively, these data suggest that G-CSF significantly augments inotuzumab-mediated ALL clearance from the bone marrow. G-CSF may provide a non-toxic and cost-effective approach to increase durable response rates to inotuzumab (and other types of immunotherapy) in patients with B ALL. [Display omitted] DisclosuresLink:Pfizer: provided Inotuzumab for preclinical studies Other.

Regulation of RAB5C is important for the growth inhibitory effects of MiR-509 in human precursor-B acute lymphoblastic leukemia.

MicroRNAs (miRs) regulate essentially all cellular processes, but few miRs are known to inhibit growth of precursor-B acute lymphoblastic leukemias (B-ALLs). We identified miR-509 via a human genome-wide gain-of-function screen for miRs that inhibit growth of the NALM6 human B-ALL cell line. MiR-509-mediated inhibition of NALM6 growth was confirmed by 3 independent assays. Enforced miR-509 expression inhibited 2 of 2 additional B-ALL cell lines tested, but not 3 non-B-ALL leukemia cell lines. MiR-509-transduced NALM6 cells had reduced numbers of actively proliferating cells and increased numbers of cells undergoing apoptosis. Using miR target prediction algorithms and a filtering strategy, RAB5C was predicted as a potentially relevant target of miR-509. Enforced miR-509 expression in NALM6 cells reduced RAB5C mRNA and protein levels, and RAB5C was demonstrated to be a direct target of miR-509. Knockdown of RAB5C in NALM6 cells recapitulated the growth inhibitory effects of miR-509. Co-expression of the RAB5C open reading frame without its 3′ untranslated region (3′UTR) blocked the growth-inhibitory effect mediated by miR-509. These findings establish RAB5C as a target of miR-509 and an important regulator of B-ALL cell growth with potential as a therapeutic target.

Abnormal expression of CD66a promotes proliferation and inhibits apoptosis of human leukemic B cells in vitro

The aberrant expression of myeloid antigen CD66 on acute B-lymphoblastic leukemia (B-ALL) cells is a well-documented phenomenon. CD66a is a major subtype of the CD66 family which plays a dual role in different cancers, and the contradictory effect may depend on the isoform ratio of CD66a-4L to CD66a-4S. Although the abnormal expression of CD66a on leukemic B-cells has been reported widely, little is known about the biological function of this aberrant expression. In this study, we showed that inhibition of CD66a in human B-ALL cell lines reduced the cellular proliferative rate and increased the percentage of cellular apoptosis, and the ratio of CD66a-4L to CD66a-4S in leukemic B cells is much higher than that in granulocytes. In addition, alteration of the L:S ratio by silencing and overexpressing the L isoform in B-ALL cell lines confirmed that a high L:S ratio of CD66a in leukemic B cells promotes proliferation and inhibits FasL-induced apoptosis.

Notch Signaling In B-ALL Reveals PLK1 As A Potential Therapeutic Target In B-ALL: PLK1-Selective Inhibitor Poloxin Modulates Cyclin B and P53 Pathways, Leading To Growth Arrest and Apoptosis

BackgroundNotch is a well-known oncogene in T-ALL, yet appears to have tumor suppressor effects in B-ALL. These cell type-specific effects of Notch signaling mirror consequences seen in early lymphocyte development and raises the question of how Notch leads to such divergent consequences in closely related cell types. In exploring these Notch mechanisms we discovered a B-ALL specific Notch-mediated reduction in the cell cycle regulator Polo-like kinase-1 (PLK1), revealing a novel targetable kinase in B-ALL. ApproachTo explore the consequences of Notch-mediated down regulation of cell cycle regulator kinase PLK1, we targeted PLK1 kinase function with the novel PLK1-selective inhibitor poloxin in human B-ALL lines. ResultsPLK1 is highly expressed in B-ALL verses normal tissues (panel A), correlates with cyclin B expression, is expressed >2-fold higher in B-ALL with t(1;19) than other B-ALL samples, and may predict response of ALL to methotrexate. In our panel of human B-ALL cell lines poloxin induced G2/M growth arrest and decreased cell number by >80% (panel B), and decreased survival in B-ALL cells (>75% AnnexinV+, panel C). PLK1 inhibition led to tumor suppressor p53 stabilization, revealing >5-fold increase in p53 protein levels following poloxin treatment in B-ALL (panel D). Mechanistically, PLK1 inhibition leads to both cytoplasmic re-localization of cyclin B, disrupting the CDC2-cyclinB complex, as well as phosphorylation of p53 at Ser20, which destabilizes p53-MDM2 interaction and thus accumulation of p53. ConclusionsWhile exploring the mechanisms of cell type-specific effects of Notch signaling in ALL, we have found a novel therapeutic target, the cell cycle regulator PLK1. Our findings reveal a novel therapeutic approach whereby PLK1-selective inhibition via poloxin induces growth arrest and apoptosis in human B-ALL via consequences on cyclin B and p53 pathways.▪ Disclosures:No relevant conflicts of interest to declare.

Next-Generation NAMPT Inhibitors For ALL Identified By Sequential High-Throughput Phenotypic Chemical and Functional Genomic Screens

There is a critical need for new agents with novel therapeutic targets and improved safety profiles in high-risk acute lymphoblastic leukemia (ALL), which is a significant cause of morbidity and mortality in pediatric and adult populations. Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of their mechanistically relevant targets remains a major experimental challenge. We applied a chemical genetics approach involving sequential unbiased high-throughput chemical and ultra-complex, genome-scale shRNA screens to address this challenge and identify novel agents in ALL. A cell-based phenotypic high-throughput chemical screen of 115,000 compounds identified 640 compounds that inhibited growth of one or both ALL cell lines with high-risk Mixed Lineage Leukemia (MLL) genetic abnormalities, but did not inhibit the growth of a cell line lacking MLL rearrangement. The most potent and selective 64 were tested on an expanded panel of eight human B-ALL cell lines to identify lead compound STF-118804. STF-118804 inhibited the growth of most B-ALL cell lines with high potency demonstrating IC50 values in the low nanomolar range. Leukemic samples from five pediatric ALL patients were also sensitive to STF-118804 in the low nanomolar range. STF-118804 displayed 5–10 fold more potency against most leukemias in comparison to cycling human (lineage-negative cord blood) and murine (c-kit+ bone marrow) progenitor cells, demonstrating a therapeutic index. STF-118804 displays distinctive cytotoxicity by inducing apoptosis without causing a phase-specific cell cycle arrest. To discover the molecular target of STF-118804, a functional genomic screen was performed to identify shRNAs that conferred sensitivity or resistance to STF-118804, utilizing an ultra-complex (∼25 shRNAs per gene) library targeting in total ∼9300 human genes and 1000s of negative control shRNAs. NAMPT was the most statistically significant gene to confer sensitivity to STF-118804, suggesting that STF-118804 functioned as a NAMPT inhibitor. NAMPT encodes nicotinamide phosphoribosyl transferase, a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a crucial cofactor in many biochemical processes. STF-118804 was confirmed as a novel class of NAMPT inhibitor through metabolic rescue, enzymatic, and genetic studies. STF-118804 displayed strong inhibitory activity in in vitro NAMPT enzymatic assays. Over-expression of wild-type or mutant NAMPT in cells indicated that STF-118804 cytotoxicity is a result of its ability to inhibit NAMPT, and that STF-118804 does not have significant off-target effects on cell viability. The potential efficacy of STF-118804 in vivo was assessed in an orthotopic xenograft model of ALL. Sublethally irradiated immunodeficient mice were transplanted with human ALL cells engineered to constitutively express firefly luciferase. Dosing of STF-118804 was initiated two weeks post-transplant when ALL cells had engrafted and bioluminescent signal was detectable. Mice treated with STF-118804 showed regression of leukemia by bioimaging and significantly extended survival. The leukemia initiating cell (LIC) frequency in STF-118804 treated mice was significantly lower (∼8 fold) than vehicle treated mice, showing that STF-118804 was effective in reducing LICs. In summary, tandem high-throughput screening identified a highly-specific, potent, and structurally novel small molecule inhibitor of NAMPT that is active in ALL. Tandem high throughput screening using chemical and ultra-complex shRNA libraries provides a rapid chemical genetics approach for seamless progression from small molecule lead identification to target discovery and validation. Disclosures:No relevant conflicts of interest to declare.

Characterization of lincRNA BALIR-6 in MLL rearranged B-lymphoblastic leukemia

A new class of non-coding RNA, known as long intergenic non-coding RNAs (lincRNAs), has only recently been described. These lincRNAs have been found to play a role in various molecular processes within the cell including gene regulation, acting as sinks for microRNAs, and regulating splicing, implicating them in development and oncogenic processes. B lymphoblastic leukemia (B acute lymphoblastic leukemia; B-ALL), a malignancy of precursor B-cells, harbors mutations and translocations that result in a dysregulated gene expression. Interestingly, dysregulated expression of lincRNAs has been found in various cancers, but has not yet been described in B-ALL. Recently, we completed a gene expression profiling study in human B-ALL samples, which showed differential lincRNA expression in samples with particular cytogenetic abnormalities. This led us to hypothesize that lincRNAs may be related to disease pathogenesis.Here, we describe a promising lincRNA from our microarray data designated B-ALL associated long intergenic RNA 6 (BALIR-6). Expression of BALIR-6 is highest in patient samples carrying the MLL rearrangement (n=16; when compared to patients with TEL-AML1-translocated, n=39; E2A-PBX1-translocated, n=8; BCR-ABL-translocated, n=3; and cytogenetically normal cases, n=56; 1-way ANOVA p<0.0001) and showed significant variance in the expression level based on the immunophenotype (1-way ANOVA p=0.0004). BALIR-6 is located on chromosome 3p24.3 in humans, and exists in a syntenic gene block in with neighboring genes SATB1 and TBC1D5, and is conserved in mammals. Rapid Amplification of cDNA Ends (RACE) uncovered multiple transcript isoforms; from these, three were cloned out and sequenced, corresponding to the genomic locus as predicted. In B-ALL cell lines, BALIR-6 expression was highest in RS411 cells, which carry the MLL rearrangement, when compared to other B-ALL cell lines. This suggests that the cell lines may show a similar expression pattern to human B-ALL samples. To study the functional role of BALIR-6 we utilized siRNA in a mmu-miR-155 expression cassette to knockdown the transcript. In RS411 cells we observed a reduction in proliferation by MTS assay. Additionally, we observed an increase Sub-G0 cells and a decrease in G2-M phase cells by propidium iodide staining, suggesting an increase in apoptosis. Conversely, overexpression of BALIR-6 in a mouse pre-B cell line (70Z/3), leads to an increase in proliferation. Interestingly, during normal B cell development, BALIR-6 is dynamically expressed, with high expression in pre-B cells and subsequent downregulation, suggesting that a normal role during development is being hijacked in patients with B-ALL.Mechanistically, a few recent studies have described that lincRNAs can regulate gene expression in cis. To explore whether BALIR 6 regulates surrounding genes in cis, we analyzed microarray data of MLL rearranged B-ALL samples, finding that expression of BALIR-6 correlates with expression of surrounding genes SATB1 and TBC1D5. Interestingly for SATB1, this correlation is also seen in human B cell developmental stages. Altering BALIR-6 expression by siRNA mediated knockdown or overexpression causes an effect on the expression of surrounding genes SATB1 and TBC1D5. Previous findings have shown that dysregulated SATB1 has been seen in a variety of malignancies, suggesting a mechanism for how BALIR-6 may produce the changes in cell growth and apoptosis described above. Altogether, these results identify a novel and interesting RNA transcript with the potential to regulate gene expression and pathogenesis in B-ALL with MLL rearrangement, suggesting novel diagnostic, prognostic, and therapeutic implications. Disclosures:No relevant conflicts of interest to declare.

CD34+CD38-CD58- Candidate Leukemia-Initiating Cells Are Clinically Relevant With The Unfavorable Prognosis In Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

▪ BackgroundThe prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) has been greatly improved in the modern era of imatinib. Nevertheless, relapse is still a major cause of treatment failure in human Ph+ALL. Leukemia-initiating cells (LICs) are presumed to be responsible for relapse in leukemia. Therefore, we conducted a study to identify the candidate LICs that are responsible for disease progression and its clinical significance in patients with Ph+ALL. AimsTo investigate the leukemia-initiating and self-renewal capacities of CD34+CD38-CD58- cells and determine the prognostic significance of CD34+CD38-CD58- phenotype in patients with Ph+ALL treated in Peking University Institute of Hematology. MethodsThe leukemia-initiating potential and self-renewal capacity of the sorted CD34+CD38-CD58-, CD34+CD38-CD58+,CD34+CD38+CD58- and CD34+CD38+CD58+ compartments were investigated in vivo using sublethally irradiated and anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice by intra-bone marrow–injection. Furthermore, we prospectively analyzed whether the identified CD34+CD38-CD58- compartment at diagnosis correlates with minimal residual disease (MRD) after therapy and clinical outcomes in 63 adult patients (18-60 years) with de novo Ph+ALL. ResultsXenotransplantation of the sorted CD34+CD38-CD58- cells led to a repopulation of human B-ALL in primary and secondary recipient mice, which were phenotypically and clonally derived from the original Ph+ALL patients analyzed by flow cytometry, as well as quantitative real-time RT-PCR and fluorescence in situ hybridization for leukemia-specific cytogenetic abnormalities. Furthermore, the candidate CD34+CD38-CD58- LICs phenotype at diagnosis (n=16) significantly correlated with a lower complete remission rate and higher MRD frequency monitored by BCR-ABL mRNA levels in BM of Ph+ALL patients. Additionally, it directly correlated with higher cumulative incidence of relapse (CIR, 60% ± 1.97% vs. 15.51% ± 0.30%, P=0.002) and unfavorable disease-free survival (DFS, 33.75%±12.64% vs. 71.31%±7.17%, P=0.009) at 3-year. The CD34+CD38-CD58- group exhibited a higher rate of BCR-ABL mutations conferring higher level imatinib resistance than the other group (43.75% vs. 17.02%, P=0.04). Multivariate analyses revealed that CD34+CD38-CD58- phenotype at diagnosis was an independent risk factor for relapse (HR=4.35, P=0.009) and DFS (HR=3.38, P=0.008) in adult Ph+ALL. Summary/ConclusionBoth the xenotransplantation data as well as the clinical correlation studies show that CD34+CD38-CD58- compartment enrich for leukemia-initiating cells in adult Ph+ALL. CD34+CD38-CD58- phenotype at diagnosis independently correlates with an adverse prognosis, which promises to be an efficient tool for relapse prediction and risk-stratification treatment in adult Ph+ALL patients. AcknowledgmentsThis work was supported by grants from National Natural Science Foundation of China (grants no. 30800483&81230013) and Beijing Municipal Science and Technology Program (grant no.Z111107067311070). Disclosures:No relevant conflicts of interest to declare.

Abstract C155: Targeting glucose metabolism to suppress Ph+B-ALL progression.

Abstract Philadelphia chromosome positive B cell derived acute lymphoblastic leukemia (Ph+B-ALL) is a type of aggressive leukemia that lacks effective treatment. The high glucose metabolism observed in many other cancers suggests that targeting glucose metabolism may provide a novel therapeutic approach for Ph+B-ALL. However, metabolic features of Ph+B-ALL and metabolic stress responses have not been described. In this study, we examined human primary B-ALL samples, B-ALL cell lines and primary murine Ph+B-ALL to identify metabolic program and metabolic stress responses of Ph+B-ALL cells. Metabolic measurements of extracellular flux and glucose consumption showed that B-ALL cell lines are highly glycolytic and preferentially utilize glucose. Consistent with a dependence on elevated glycolysis, primary human B-ALL cells were more sensitive than normal B cells to inhibition of glycolysis with 2-deoxyglucose (2-DG). This metabolic stress induced cell death was likely to be mediated through p53 and Bcl-2 family proteins. 2-DG treatment induced expression of pro-apoptotic Bcl-2 family protein Bim and primary murine Ph+B-ALL cells generated from p53-/- or Bim-/- background remained viable even when treated with 2-DG. To specifically investigate how inhibition of glucose metabolism impacts B-ALL survival and disease progression in vivo with genetic tools, we generated murine Ph+ B-ALL cells on a Glut1fl/fl Ubi-CreER background that allow specific deletion of Glut1 in cancer cells upon treatment of tamoxifen. In vitro deletion of Glut1 reduced glucose uptake and glucose metabolism, although glycolysis was not wholly suppressed. This partial reduction of glucose metabolism led to greatly reduced cell proliferation and some cell death in vitro. Importantly, in vivo deletion of Glut1 after transfer of B-ALL cells into congenic recipients suppressed B-ALL progression and prolonged animal survival. These data show that B-ALL cells exhibit high glucose metabolism similar to other types of cancer and are sensitive to glucose metabolism inhibition. Reduction of glucose metabolism can impede B-ALL proliferation or cause cell death through p53 and Bim and thus suppress B-ALL progression in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C155. Citation Format: Tingyu Liu, Handan Xiang, Amanda Nichols, Valerie Gerriets, Rigel Kishton, David Rizzieri, Jeffrey C. Rathmell. Targeting glucose metabolism to suppress Ph+B-ALL progression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C155.

Abstract 5027: In vivo effects of TSLP in a human-mouse xenograft model of CRLF2 B-ALL.

Abstract Pediatric CRLF2 B-cell Acute Lymphoblastic Leukemia (CRLF2 B-ALL) is a high-risk form of leukemia that is associated with relapse and poor patient survival outcomes. This leukemia occurs five times more frequently among children of Hispanic/Latino ethnicity than others and represents the most significant biological component of childhood cancer health disparities identified to date. CRLF2 B-ALL arises from genetic alterations that result in overexpression of the CRLF2 gene. CRLF2, together with the IL-7 receptor α, forms a receptor complex that is activated by the cytokine, TSLP. The JAK-STAT5 pathway is phosphorylated downstream of receptor activation. The activating JAK mutations found in some CRLF2 B-ALL have led to speculation that TSLP stimulation is not a factor in CRLF B-ALL. However, we found that TSLP increases STAT5 phosphorylation, even in CRLF2 B-ALL cells with JAK mutations. Our studies of normal B cell development show that TSLP stimulation increases cellular proliferation in early human B lineage cells. We also find that pediatric bone marrow (BM) stromal cells express TSLP and thus provide an in vivo source of TSLP that can act on CRLF2 B-ALL cells. We proposed to evaluate the role of TSLP in human CRLF2 B-ALL using a human-mouse xenograft model. However, TSLP is different from most other cytokines produced in the mouse in that it is species-specific and therefore does not activate the human TSLP receptor complex that contains CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that we believe to be a contributing factor in CRLF2 B-ALL. To overcome this hurdle we engineered immune deficient mice to express human TSLP (hTSLP+ mice) as well as control mice that lack the human TSLP cytokine (hTSLP- mice). We used this hTSLP+/- xenograft model system to evaluate the in vivo effects of TSLP on survival and proliferation of transplanted CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line). Mice were euthanized at 5 weeks and BM was harvested. Evaluation of BM disease by flow cytometry showed that the percentage of viable human leukemia cells in hTSLP+ mice was twice that observed in hTSLP-mice. Evaluation of cell cycle progression in human CRLF2 B-ALL cells isolated from xenograft BM showed that the percentage of cycling cells in hTSLP+ mice was 2.5 fold higher than in hTSLP- mice. These preliminary data suggest that TSLP may contribute to CRLF2 B-ALL by increasing the survival and proliferation of CRLF2-B-ALL cells. Ongoing studies are focused on evaluating in vivo effects of TSLP in primary CRLF2 B-ALL from Hispanic patients. The identification of genes downstream of TSLP-CRLF2 signaling has the potential of providing drug targets for combination therapy to effectively treat CRLF2 B-ALL and reduce cancer health disparities in Hispanic childhood B-ALL. This work is supported by NIH R21R21CA162259, a St. Baldrick's Foundation Research Grant, and a LLU GCAT award (KJP) Citation Format: Rui-jun Su, Olivia L. Francis, Shannalee R. Martinez, Ineavely Baez, Terry-Ann Milford, Christopher L. Morris, Ross O. Fisher, Xiao-Bing Zhang, Sinisa Dovat, Kimberly J. Payne. In vivo effects of TSLP in a human-mouse xenograft model of CRLF2 B-ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5027. doi:10.1158/1538-7445.AM2013-5027

Abstract 4612: Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth.

Abstract The current understanding of the Warburg effect consists of an increase in aerobic glycolysis in cancer cells. The connection between glycolysis and PPP/biosynthesis is based upon a model in which glycolytic intermediates can be diverted into PPP and biosynthesis pathways as precursors. However, it remains unclear how cancer cells coordinate glycolysis and biosynthesis to fulfill the request of rapidly growing tumors. We found that glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). Our co-crystal structure based analysis revealed that 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP) as a competitive inhibitor, while 2-PG activates 3-phosphoglycerate dehydrogenase (PHGDH) to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA results in increased 3-PG and decreased 2-PG levels in cancer cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth. These findings uncover that PGAM1 controls the intracellular 3-PG and 2-PG levels to serve as a novel link between glycolysis, biosynthesis, cancer cell proliferation and tumor development. Moreover, we screened and developed novel small molecule PGAM1 inhibitor (PGMI)-004A, which effectively inhibits PGAM1 enzyme activity, resulting in aberrant cancer cell metabolism with reduced glycolysis and PPP/anabolic biosynthesis, as well as attenuated cell proliferation in diverse human cancer cell lines, but not control normal human proliferating cells including human dermal fibroblasts and human foreskin fibroblasts. PGMI-004A treatment attenuates tumor growth in xenograft nude mice with minimal toxicity in vivo. Furthermore, PGMI-004A inhibits cell proliferation of primary leukemia cells from human AML, CML and B-ALL patients, but not control CD34+ cells isolated from bone marrow samples or mononucleocytes isolated from peripheral blood samples from healthy donors, suggesting minimal toxicity of PGMI-004A in human cells.These results together provide “proof of principle” for the development of PGAM1 inhibitors as anti-cancer agents. Citation Format: Taro Hitosugi, Lu Zhou, Martha Arellano, Hanna Khoury, Titus Boggon, Sumin Kang, Chuan He, Jing Chen. Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4612. doi:10.1158/1538-7445.AM2013-4612

Abstract SY05-01: Genetic and non-genetic mechanisms contribute to long-term clonal growth dynamics and therapy resistance.

Abstract SY05-01: Genetic and non-genetic mechanisms contribute to long-term clonal growth dynamics and therapy resistance.

Phosphoglycerate Mutase 1 Promotes Leukemia Metabolism by Coordinating Glycolysis and Biosynthesis, and Represnts a Therapuetic Target in Leukemia Treatment

AbstractAbstract 860The current understanding of the Warburg effect consists of an increase in aerobic glycolysis in cancer cells. The connection between glycolysis and PPP/biosynthesis is based upon a model in which glycolytic intermediates can be diverted into PPP and biosynthesis pathways as precursors. However, it remains unclear how cancer cells coordinate glycolysis and biosynthesis to fulfill the request of rapidly growing tumors. We found that glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers including leukemias due to loss of TP53, regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). Our co-crystal structure based analysis revealed that 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP) as a competitive inhibitor, while 2-PG activates 3-phosphoglycerate dehydrogenase (PHGDH) to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA results in increased 3-PG and decreased 2-PG levels in cancer and leukemia cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth. These findings uncover that PGAM1 controls the intracellular 3-PG and 2-PG levels to serve as a novel link between glycolysis, biosynthesis, cancer/leukemia cell proliferation and tumor development.Moreover, we found that PGAM1 protein expression and enzyme activity levels are commonly upregulated in diverse human leukemia cell lines, as well as in primary leukemia cells from human AML, CML and B-ALL patients. We screened and developed novel small molecule PGAM1 inhibitor (PGMI)-004A, which effectively inhibits PGAM1 enzyme activity, resulting in aberrant leukemia cell metabolism with reduced glycolysis and PPP/anabolic biosynthesis, as well as attenuated cell proliferation in diverse human leukemia cell lines, but not control normal human proliferating cells including human dermal fibroblasts and human foreskin fibroblasts. In addition, PGMI-004A treatment attenuates tumor growth in xenograft nude mice with minimal toxicity in vivo. Furthermore, PGMI-004A inhibits cell proliferation of primary leukemia cells from human AML, CML and B-ALL patients, but not control CD34+ cells isolated from bone marrow samples or mononucleocytes isolated from peripheral blood samples from healthy donors, suggesting minimal toxicity of PGMI-004A in human cells.Together, our findings demonstrate that PGAM1 regulates the concentrations of glycolytic metabolites 3-PG and 2-PG, which function as signaling molecules to directly affect the catalytic activity of enzymes involved in PPP and biosynthesis, representing a novel, additional link between glycolysis, PPP and biosynthesis. PGAM1 is commonly upregulated in human leukemia cells and important for cell metabolism and proliferation, representing a promising therapeutic target in treatment of leukemias. Small molecule inhibitor of PGAM1, PGMI-004A exhibits promising efficacy and minimal toxicity in treatment of xenograft nude mice and human primary leukemia cells, providing “proof of principle” for the development of PGAM1 inhibitors as anti-leukemia agents. Disclosures:No relevant conflicts of interest to declare.