Patient-derived ALL Cells Research Articles

Development of Potent and Selective Janus Kinase 2/3 Directing PG-PROTACs.

Aberrant activation of the JAK-STAT signaling pathway has been implicated in the pathogenesis of a range of hematological malignancies and autoimmune disorders. Here we describe the design, synthesis, and characterization of JAK2/3 PROTACs utilizing a phenyl glutarimide (PG) ligand as the cereblon (CRBN) recruiter. SJ10542 displayed high selectivity over GSPT1 and other members of the JAK family and potency in patient-derived ALL cells containing both JAK2 fusions and CRLF2 rearrangements.

Reciprocal Regulation of BCL6 and MLL Expression As a Vulnerability in MLL -Rearranged Acute Lymphoblastic Leukemia

Background: Chromosomal translocations involving the Mixed Lineage Leukemia gene MLL account for ~70% of infant acute lymphoblastic leukemias (ALLs; Ayton and Cleary, 2001) and are associated with dismal prognosis. These findings underscore the importance of understanding the molecular mechanisms underlying disease progression of MLL-rearranged (MLLr) leukemia for the development of novel therapeutics. Recently, an integrative epigenomic analysis has identified the transcriptional repressor and proto-oncogene BCL6 as a transcriptional target in MLL-AF4 -driven pre-B ALL (Geng et al., 2012). BCL6 functions as a mediator of drug resistance to tyrosine kinase inhibitors in Ph+ ALL (Duy et al., 2011). Here, we tested the hypothesis that BCL6 is required for MLLr leukemia and represents a therapeutic target in MLLr pre-B ALL.Results: Western blot analysis of a panel of patient-derived pre-B ALL cells (MLLr : n= 7; ETV6-RUNX1 : n= 2; BCR-ABL1 : n= 1; not otherwise specified: n= 4) and normal CD19+ B cells (n= 3) revealed that BCL6 levels were elevated in MLL r ALL samples. Furthermore, immunohistochemistry staining in bone marrow biopsies from ALL patients (n= 72) showed that BCL6 levels were upregulated in MLLr cases. Expression of MLL-ENL or MLL-AF4 fusion protein increased Bcl6 protein levels (~10-fold) in murine pre-B cells. Moreover, ChIP-seq analysis of human MLLr pre-B ALL cell lines using antibodies against MLL, AF4 and ENL demonstrated direct binding to the BCL6 promoter. Studying methylation status revealed that the BCL6 promoter was hypomethylated in patients with MLLr pre-B ALL compared to patients with other subtypes of pre-B ALL (including BCR-ABL1, ETV6-RUNX 1 and hyperdiploid) as well as normal pre-B cells from healthy donors (St. Jude, n= 132, Figueroa, 2014). Consistent with these observations, multivariate analysis of high risk pre-B ALL patients showed that high BCL6 levels in combination with MLL rearrangements were associated with the worst clinical outcome (COG P9906). Collectively, our findings demonstrated that BCL6 levels are elevated in MLLr ALL, and that expression levels of MLL and BCL6 can serve as predictors for risk stratification in ALL.Interestingly, expression of wild-type MLL was positively correlated with that of BCL6 in both pediatric (COG P9906) and adult (ECOG E2993) pre-B ALL. Furthermore, multivariate analysis comparing MLLHighBCL6High patients with MLLLowBCL6Low patients showed that high expression levels of both genes correlated with poor clinical outcome (COG P9906). These findings prompted us to study whether MLL regulates BCL6 expression, and vice versa . Cre-mediated deletion of Mll abrogated imatinib-mediated induction of Bcl6 in BCR-ABL1 -driven pre-B ALL, indicating that Mll is required for upregulation of Bcl6 expression. While genetic inactivation of Bcl6 as well as treatment with the BCL6-specific retro-inverso peptide-inhibitor (RI-BPI) decreased Mll expression, doxycycline-induced expression of Bcl6 upregulated Mll expression. Combining chromatin immunoprecipitation and DNA microarray (ChIP-on-chip), we found that BCL6 was recruited to the MLL promoter in pre-B ALL. Taken together, these findings support our proposed scenario of reciprocal regulation of MLL and BCL6 expression.Notably, ChIP-seq analyses of MLLr pre-B ALL cells showed that there are 268 common gene targets of both MLL and BCL6. Among the common targets include cell cycle checkpoint regulators (CDKN1B, ETS2, BMI1, RB1), apoptosis facilitator BIM, and tumor suppressor TET2, indicating that BCL6 is a key regulator of the transcriptional program in MLLr pre-B ALL. In support of an essential role of BCL6 in MLLr ALL, expression of a dominant negative BCL6 mutant in patient-derived MLLr pre-B ALL cells induced rapid depletion from cell culture in competitive-growth assays. Furthermore, RI-BPI treatment resulted in cell cycle arrest, impaired colony forming ability and inhibited leukemia initiation in transplant recipient mice. Importantly, RI-BPI in combination with standard chemotherapy Vincristine exerted more potent effects on killing MLLr pre-B ALL cells than either agent alone.Conclusions: Our findings suggest that aberrant BCL6 expression levels are important for disease progression, and that pharmacological inhibition of BCL6 augments responsiveness to standard chemotherapy in the treatment of MLLr pre-B ALL. DisclosuresHurtz:Incyte: Research Funding. Armstrong:Cyteir: Consultancy; Imago: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

PON2 Exemplifies a Unique Dependency of B Cell Lineage ALL Cells on Detoxifying Lactonases

Background and significance: Paraoxonase 2 (PON2) is a member of mammalian detoxifying enzymes that are located to the mitochondrial membrane, hydrolyze lactone metabolites and interact with coenzyme Q10 to diminish oxidative stress. While PON2 is highly expressed in the CNS and multiple fetal tissues, expression levels in normal hematopoietic cells are low. We began to study the function of PON2 in B cell lineage ALL, because microarray analyses suggested high mRNA levels of PON2 in B cell lineage ALL cells. In addition, PON2 is used as a diagnostic marker on a 15 gene diagnostic LDA panel (e.g. in AALL1131; NCT02883049) that is used for the identification of Ph-like ALL, a subgroup with particularly poor outcome and specific treatment requirements.Results: Analyzing data from pediatric and adult clinical trials, we found that greater than median PON2 mRNA levels at the time of diagnosis predicted poor clinical outcomes for both children (COG P9906; n=207; P=1.09e-05) and adults (ECOG; n=215; P=0.003) with B-lineage ALL. Studying expression levels of PON2 by quantitative RT-PCR and Western blot in normal bone marrow B cell precursors from healthy donors and patient-derived pre-B ALL cells including Ph+ and Ph -like ALL, we found 3-10-fold increased mRNA and protein levels of PON2 throughout multiple pre-B ALL samples. To elucidate potential functions of PON2 in normal B cells and B-lineage ALL, we studied normal B cell development and ALL-models in Pon2-/- mice. While B cell development was unperturbed in Pon2 -deficient mice, deletion of Pon2 had profound effects on both BCR-ABL1- and N RASG12D-driven leukemogenesis. Compared to wildtype, Pon2-/- pre-B ALL cells failed to form colonies in semisolid agar. Pon2-/-ALL cells were arrested in G0/G1 phase and expressed 2-5-fold increased levels of Arf and p21, compared to Pon2+/+ ALL cells. Strikingly, >50% of Pon2-/-ALL cells spontaneously underwent cellular senescence, as shown by b-galactosidase staining in conjunction with increased Arf expression levels. These in vitro findings suggest an important role of PON2 in B-lymphoid leukemogenesis, which was confirmed in transplant experiments based on BCR-ABL1 and N RASG12D ALL models. While Pon2 -deficiency substantially prolonged survival of recipient mice of BCR-ABL1 ALL cells (P=0.0001), mice transplanted with Pon2 -deficient N RASG12D ALL cells survived for indefinite periods of time.Targeting PON2 expression in relapse ALL: Given that PON2 is expressed at very high levels in relapse ALL samples, we tested the concept of targeting PON2 lactonase activity in a prodrug-approach. While PON2 activity typically results in detoxification of lactone-metabolites, lactone-hydrolysis of the N-(3-oxododecanoyl)-homoserine lactone (3OC12) prodrug results in cytotoxic byproducts (Guoping et al., 2016). We therefore tested the therapeutic potential of 3OC12. For genetic validation, we treated wildtype and Pon2-/- ALL cells with 3OC12 and found strong cytotoxic effects in wildtype but not Pon2-/- ALL cells. Likewise, inducible overexpression of Pon2 in patient-derived pre-B ALL cells exacerbated toxicity of 3OC12 compared to empty vector controls. Likewise, CRISPR-Cas9 mediated ablation of PON2 in human ALL PDX reversed sensitivity of the pre-B ALL cells to 3OC12.Conclusion: Here we describe the previously unknown function of the detoxifying PON2 lactonase as an essential prerequisite for pre-B cell transformation and leukemogenesis. PON2 expression is specific for leukemia cells, an outcome predictor for patients with pre-B ALL and a biomarker of Ph-like ALL. While PON2 protects ALL cells and enables malignant growth, we demonstrate that its lactonase-activity can be leveraged for pharmacological targeting as exemplified by the lactone-prodrug 3OC12. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional Control of Glucose and Energy Supply Prevents Oncogenic Signaling and B Cell Transformation

Background and Hypothesis: B-cell identity is determined by a set of B-cell transcription factors including PAX5, IKZF1 and EBF1. However, B-lineage leukemia clones often carry secondary genetic lesions that result in reduced activity or inactivation of these transcription factors. Studying patient samples from clinical trials for B-lineage childhood (P9906; n=187) and adult (MDACC; n=92) leukemia, we found that genetic defects in one or more B-cell transcription factors represent near-obligate lesions in human acute lymphoblastic leukemia (209 of 279 B-lineage ALL cases). While previously of unknown significance, we found that adult ALL cases with known lesions in one or more of these transcription factors had higher activity of lactate dehydrogenase, and phospho-states indicated higher activity of IRS1, PDK1, and AKT, which contribute to glucose uptake. For this reason, we investigated whether B-cell transcription factors set metabolic constraints of oncogenic signaling in leukemia.Results: Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells carrying PAX5 and IKZF1deletions decreased phospho-AKT levels. Furthermore, protein levels of multiple positive regulators involved in glucose uptake and metabolism (including insulin receptor, glucose transporters and hexokinases) were downregulated upon reconstitution of PAX5 and IKZF1. Conversely, protein levels of negative regulators of glucose uptake and metabolism including NR3C1 and TXNIP were upregulated upon reinstatement of PAX5 and IKZF1 function. Analysis of ChIPseq data of human B cells revealed binding of multiple B-cell transcription factors including PAX5, IKZF1 and EBF1 to promoter regions of genes encoding positive regulators of glucose uptake and metabolism. Binding peaks for B-cell transcription factors were also observed at genes that encode negative regulators of glucose uptake and metabolism (NR3C1 and TXNIP). In addition, direct recruitment of PAX5 was confirmed by single-locus quantitative chromatin immunoprecipitation (qChIP).Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells caused depletion from the cell culture in competitive growth assays, in parallel with reduced glucose consumption and depletion of cellular ATP levels. On the contrary, overexpression of dominant-negative PAX5-ETV6 and IK6 in patient-derived B-lineage ALL cells expressing wildtype PAX5 and IKZF1 resulted in a net survival advantage, concomitant with increases in both glycolytic activity and cellular ATP levels. PAX5-mediated impaired survival fitness was significantly rescued by CRISPR-based activation of gene expression of insulin receptor, glucose transporter and hexokinases in patient-derived B-lineage ALL cells. Conversely, CRISPR/Cas9-mediated deletion of NR3C1 and TXNIP largely reversed the effects of PAX5. Finally, reduced survival fitness upon reconstitution of wild-type PAX5 and IKZF1 in patient-derived pre-B ALL cells was mostly rescued by metabolites that can enter the TCA cycle and thus provide ATP.Conclusions: In summary,B-cell-specific restriction of glycolytic energy supply represents a previously unrecognized metabolic barrier against malignant transformation. Our findings suggest a causative link between impaired glucose uptake and metabolism caused by B-cell transcription factors as well as cell death, and the known tumor suppressive function of PAX5 and IKZF1 in B-lineage ALL. DisclosuresNo relevant conflicts of interest to declare.

Phloridzin Docosahexaenoate (PZ-DHA) : A Novel Naturally Derived Drug Active in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant disease that accounts for about 15% of pediatric and 25% of adult ALL. Although risk stratification has provided more tailored therapy and improved the overall survival of T-ALL patients, clinical challenges such as suboptimal drug responses, morbidity from drug toxicities, and drug resistance still exist. Plant polyphenols have therapeutic efficacy as pharmacological adjuvants to help overcome these challenges. They can be acylated with fatty acids to overcome issues concerning bioavailability, such as poor intestinal absorption and low metabolic stability. Phloridzin (PZ), a flavonoid found in apple peels, was acylated with an omega-3 fatty acid, docosahexaenoic acid (DHA), to generate a novel ester called phloridzin docosahexaenoate (PZ-DHA). The cytotoxic effect of PZ-DHA was studied in the human Jurkat T-ALL cell line. PZ-DHA significantly reduced the viability and cellular ATP levels of treated cells. PZ-DHA was found to selectively induce apoptosis in Jurkat cells, while sparing normal murine T-cells. Apoptosis was further confirmed by demonstrating the ability of PZ-DHA to induce morphological alterations, DNA fragmentation, caspase activation, and the release of intracellular lactate dehydrogenase. PZ-DHA also significantly inhibited cell division in Jurkat cells. Furthermore, interferon-α-induced phosphorylation of the transcription factor, STAT3, was downregulated following PZ-DHA treatment. The in vitro efficacy of PZ-DHA was recapitulated in vivo in an established zebrafish xenograft model, where the proliferation of transplanted Jurkat cells was inhibited when PZ-DHA was added to the embryo water. Overall, these findings provide evidence for PZ-DHA as a novel therapeutic agent with activity in T-ALL. Studies examining the effect of PZ-DHA on patient-derived ALL cells engrafted in zebrafish are currently underway. DisclosuresNo relevant conflicts of interest to declare.

Overcoming Drug Resistance of Pre-B ALL Cells By Targeting Integrin alpha6 Associated Cell-Adhesion Mediated Drug Resistance Using a Novel Antibody, P5G10

Background. The presence of chemotherapy-resistant cells can be detected in the bone marrow (BM) and peripheral blood (PB) and is called minimal residual disease (MRD). The exact mechanism for cell adhesion-mediated drug resistance leading to MRD and how to therapeutically target MRD is unsolved.Integrin alpha-6 (alpha6) is expressed on normal hematopoietic cells but has recently been described as a novel marker for MRD+ ALL cells. We previously described a role of alpha6 as a critical molecule in drug resistance of ALL. Here we extend our studies evaluating a novel non-humanized antibody targeting alpha6, P5G10, as a novel therapy against drug resistant ALL.Method. For in vitro studies patient-derived (primary) pre-B ALL cells co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells or counter-ligand laminin-1. Annexin V/7-AAD staining was used for viability determination by flow cytometry. A NOD/SCID IL2Rγ-/- (NSG) xenograft model of primary pre-B ALL was used for in vivo experiments.Results. We evaluated integrin alpha6 blockade in four primary ALL cells (LAX7R, PDX2, TXL3, SFO2) using an anti-functional alpha6 antibody, P5G10, with and without the counter ligand laminin-1 or OP9. Alpha6 blockade de-adhered all four cases from laminin-1 compared to control-treated cells and percentage of adherence was significantly different (3.3%±0.6% vs. 77.7%±3.3%, p= 0.0002 for LAX7R; 10.5%±4.9% vs. 72.5%±0.7%, p= 0.003 for PDX2; 2.0%±1.3% vs. 66.9.6%±2.6%, p=0.0002 for TXL3; 9.6%±2.8% vs. 68.0%±5.7%, p=0.0006 for SFO2).. P5G10 de-adhered leukemia cells to a lesser degree from OP9-coated plates indicating that other adhesion molecules also contribute to leukemia cell adhesion. To determine the effect of alpha6 modulation in chemoresistant ALL, primary BCR-ABL1- ALL cells (LAX7R, SFO2) were treated with Vincristine, Dexamethasone and L-Asparaginase (VDL) and BCR-ABL1+ ALL cells (TXL3, PDX2) were treated with a tyrosine kinase inhibitor (TKI), Nilotinib (NTB). Primary ALL cells showed decreased viability after monotreatment with P5G10 in a short-term assay of 2 days with laminin-1 and were sensitized when P5G10 was combined with VDL or TKI, compared to TKI monotreatment (Cell viabilities were as follows: LAX7R, 13.9%±0.6% vs. 28.8%±2.6%, p=0.009; PDX2, 12.7%±1.4% vs. 19.9%±1.5%, p= 0.037: TXL3, 32.9%±2.6% vs. 48.3%±2.5%, p=0.026; and SFO2, 34.4%±7.9% vs. 47.6%±0.1%, p=0.047). Critically, in a long-term co-culture assay of primary ALL cells with OP9 cells, alpha6 blockade in combination with VDL or NTB lead to marked decrease in viability of ALL cells compared to VDL or NTB treatment (26.5%±10.0% vs. 74.2%±2.7%, p=0.002 for LAX7R and 33.5%±11.4% vs. 84.9%±15.1%, p=0.031 for SFO2 on day 17 post treatment, respectively). To determine if P5G10 induces mobilization of leukemia cells to the peripheral blood, patient-derived ALL cells, 3 cases (TXL3, PDX2 and LAX7R) were injected into NSG mice. After determination of engraftment of leukemia by flow cytometry of human CD45 in the PB, recipient mice were treated with 30mg/kg P5G10 or PBS control by i.v. or i.p. injection. The % of human CD45+ and CD19+ in peripheral blood (PB) was analyzed by flow cytometry before (pre), 1 and 3 days after (post) treatment with P5G10. In all 3 cases, we did not observe an increase of leukemia cells in the PB compared to before P5G10 treatment (Day 0) or compared to the control recipient mice. Critically, we determined, if P5G10 can restore chemosensitivity of leukemia cells in vivo. For this purpose, we injected luciferase-labeled LAX7R cells into NOD/SCID mice. Three days after leukemia cell injection leukemia cell-bearing mice received four weekly injections of 30mg/kg P5G10 or saline ± VDL. Mice treated with P5G10 survived similarly as untreated mice (PBS: MST = 39 days vs. P5G10: MST = 31 days; p=0.05). In marked contrast, mice treated with VDL plus P5G10 survived disease-free compared to chemotherapy-only treated mice until the experiment was terminated Day 186 post-leukemia injection (MST= 185 days vs. MST=71 days; p=0.0012). Human CD19 or CD45 was undetectable in the peripheral blood by flow cytometry in surviving P5G10+VDL-treated animals before they were sacrificed.Conclusion. Taken together, we demonstrate that alpha6 may be a novel therapeutic target in ALL and modulating the function of integrin alpha6 using P5G10 can overcome drug resistance in ALL. DisclosuresNo relevant conflicts of interest to declare.

IFITM3 (CD225) Links the B Cell Antigen CD19 to PI3K-AKT Signaling in Human ALL Cells

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21. Recent immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013). However, in some cases, CART19 treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Therefore, we studied factors that regulate CD19 surface expression on human pre-B ALL cells.Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and activation of checkpoint molecules p53 and p21, reduction of BCL2 and BCLXL levels and increased propensity to apoptosis. Reconstitution of IFITM3 reversed these changes.Conversely, forced expression of IFITM3 in patient-derived pre-B ALL cells increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Although human IFITM1 has known as a component of B cell receptor complex, co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT. In addition, agonistic antibodies against IFITM3/CD225 trigger CD19/PI3K-AKT signaling, which caused massively increased proliferation of pre-B ALL cells. Interestingly, agonistic antibodies against CD19 had no effect on the proliferation of pre-B ALL cells despite their ability to activate CD19/PI3K-AKT signaling. These findings indicate a specific role of IFITM3 in regulating CD19/PI3K-AKT signaling in malignant pre-B ALL cells compared to their normal pre-B cell counterparts.Conclusion: These findings identify novel role of the IFITM3 (CD255) surface receptor in regulating CD19 phosphorylation and CD19-Y513 mediated PI3K-AKT signaling in pre-B ALL cells. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K-AKT in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

The PI3Kdelta Inhibitor, CAL-101, Inhibits Migration of Primary Pre-B ALL to SDF1alpha: Treatment Implications for Overcoming Cell-Adhesion-Mediated Drug Resistance

BACKGROUND: ALL cells are physically anchored in the bone marrow (BM) microenvironment through a network of adhesion molecules. Adhesion also creates intracellular signals that regulate proliferation and cell death. We have previously identified integrin α4 (α4) as a critical molecule for such cell-adhesion-mediated drug resistance in pre-B ALL. In chronic lymphocytic leukemia (CLL), α4-mediated activation of the PI3K/AKT pathway was reported. In ALL, stromal cell protection of B-lineage ALL cells has also been shown to require active Akt. In addition, we have shown that treatment with CAL-101, a commercially available PI3Kδ inhibitor, de-adheres pre-B ALL cells from the α4-counter-receptor VCAM1, downregulates p-Akt and induces apoptosis in primary pre-B ALL cells. However, the microenvironmental stimuli that activate PI3K/AKT in ALL remain unknown. PI3Kδ can be activated by extracellular signals upon engagement with extracellular matrix, however specific ligands remain elusive, the identification of which would be critical to understand the mechanism of PI3Kδ inhibition. These studies were designed to determine which extracellular ligands activate PI3K/AKT in patient-derived (primary) pre-B ALL cells and whether CAL-101 affects adhesion and migration of ALL cells.METHODS: Four samples ofpatient-derived (primary) pre-B ALL cells, co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells were treated with a commercially available PI3Kδ inhibitor, CAL-101 (Selleckchem), or its vehicle DMSO control. Culture plates with and without transwells were used for in vitro assays. Annexin V/7-AAD staining was used for viability determination by flow cytometry and Western Blot was used for determination of total Akt and p-Akt expression.RESULTS: To determine which ligands activate p-Akt, ALL cells were adhered to fibronectin (FN), VCAM-1 (both counter-receptors of integrin α4), laminin-1 (hLam-1; counter-receptor for integrin α6) and albumin (BSA). OP9 cells were used in direct contact with ALL cells or separated through a transwell insert. α-MEM or RPMI were used as media controls. hVCAM-1 and hLam-1 activated p-Akt in one out of three samples. In all 3 samples, OP9 cells activated p-Akt compared to media control, but only in direct contact, not when separated through a transwell. This suggests that direct adhesion of ALL cells to stromal cells rather than diffusible factors are associated with p-Akt activation. Next, we determined if CAL-101 de-adheres ALL cells independently from the ligands ALL cells are bound to. Primary pre-B ALL cells were plated in triplicates onto FN, VCAM-1, hLam1-coated 96-well plates and incubated with CAL-101 (10µM) ± HUTS-21 antibody (20µg/mL). HUTS-21 is an antibody that binds only to the active form of integrin β1, CD29, and can activate outside-in signaling via integrin β1 stimulation. CAL-101 inhibited cell-adhesion from FN (15±4.2% vs. 51±9.9%, p=0.04) and VCAM-1 (54.6 x±8.6%vs. 95.8±3.8%, p=0.03), but not from hLam1 indicating that it acts specifically on the VCAM-1/FN-mediated adhesion of ALL cells. HUTS-21 restored cell-adhesion of ALL cells to FN (67.0±1.4% vs. 81±7.1%, p=0.19) and VCAM-1 (42.5±16.2% vs. 50.6±8.6%, p=0.59) indicating adhesion recovering upon HUTS-21 addition to CAL-101-treated cells. Next, we determined if CAL-101 has an impact on migration of ALL cells. ALL cells were treated with CAL-101 (10μM) or DMSO control for 24 hours and added to a migration assay. We observed that migration of ALL cells to stromal cell-derived factor 1alpha (SDF-1alpha; CXCL12) was significantly decreased (1.7 x103 ±0.3x103 vs. 3.1x103 ±2.4x103 p=0.004), while migration to OP9 cells was partially inhibited compared to DMSO control (2.1 x103 ±0.5x103 vs. 4.2 x103 ±0.5x103 p=0.005).CONCLUSION: Taken together, our data demonstrate that the PI3Kδ/AKT pathway is stimulated by extracellular matrices and that PI3Kδ inhibition with CAL-101 inhibits adhesion of ALL cells to FN/VCAM-1 but not Lam1, which can be restored using HUTS-21. Data derived from further studies will determine the potential of the FDA-approved PI3Kδinhibitor Idelalisib as a novel therapy for pre-B ALL. DisclosuresWayne:NIH: Patents & Royalties; Medimmune: Honoraria, Other: travel support, Research Funding; Kite Pharma: Honoraria, Other: travel support; Pfizer: Honoraria; Spectrum Pharmaceuticals: Honoraria, Other: travel support, Research Funding.

CD25 (IL2RA) Orchestrates Negative Feedback Control and Stabilizes Oncogenic Signaling Strength in Acute Lymphoblastic Leukemia

Background and hypothesis: CD25 (IL2RA) represents the α chain of the interleukin 2 receptor on T cells and plays an important role in the maintenance of regulatory T (Treg) cells, hence preventing T cell autoimmunity. In a comprehensive gene expression analysis, we found that CD25 is specifically upregulated by pre-B cell receptor (pre-BCR) signaling during early B cell development and oncogenic tyrosine kinase that mimic pre-BCR signaling (e.g. in Ph+ ALL and Ph-like ALL). In adults with Ph+ ALL (ECOG; MDACC) and children with Ph-like ALL (P9906) patients with CD25 expression at the time of diagnosis have a particularly poor outcome (n=416; P =0.005). For these reasons, we studied the function of CD25 in B cell development and leukemia in a series of genetic experiments.Results: Unlike T cells, CD25 (IL2RA) does not function as IL2 receptor chain in B cells and B-lineage ALL: CD25 expressed on B-lineage cells did not pair with IL2Rb and g-chains and was not responsive to IL2. Il2ra-/- B cells were arrested at the pre-B cell stage with hyperactive pre-BCR downstream signaling including SRC, BTK and ERK. In the presence of CD25, Il2ra+/+ B cells responded to engagement of the pre-BCR with phosphorylation of pre-BCR downstream tyrosine kinases and coordinated release of Ca2+ from cytoplasmic stores. In the absence of CD25 (Il2ra-/-), the pre-BCR signals autonomously, resulting in uncoordinated Ca2+ oscillations of variable duration. While CD25 does not function as IL2 receptor chain in B cells, it coordinates pre-BCR-dependent signal transduction and regulates its intensity.The pre-BCR related tyrosine kinase BTK is phosphorylated by BCR-ABL1 in Ph+ ALL and other tyrosine kinase oncogenes in Ph-like ALL (Chen et al., 2015). Interestingly, overexpression of a constitutively active form of BTK resulted in strong upregulation of CD25 surface expression. Conversely, the BTK-inhibitor ibrutinib abolished CD25 expression suggesting that feedback control between pre-BCR signaling and CD25 requires BTK.The ability of CD25 to stabilize oncogenic signaling strength in Ph+ ALL and Ph-like ALL was important for leukemia-initiation and development of fatal disease. In the absence of CD25, Il2ra-/- ALL cells showed impaired proliferation and colony formation. Serial transplantation experiments revealed a profound defect of Il2ra-/- ALL cells to initiate leukemia. 100-times more cells were required to cause fatal disease. In addition, CD25 expression mediated drug-resistance in ALL cells: In patient-derived pre-B ALL cells with heterogeneous CD25 expression, vincristine selectively induced apoptosis in CD25Low cells but spared CD25High ALL cells. Combination with an anti-CD25 immunotoxin efficiently eradiated CD25High leukemia cells and sensitized the ALL cell population to treatment with vincristine.To elucidate the mechanism of how CD25 coordinates negative feedback control of pre-BCR signaling or its oncogenic mimics, we focused on its short (13aa) cytoplasmic tail, which includes two phosphorylation sites (S268 and T271) that are known substrates for serine/threonine protein kinase, PKCα. To identify cytoplasmic interaction partners of CD25, we overexpressed a Flag-tagged truncated form of CD25 including a myristoylation signal for constitutive membrane localization, transmembrane domain and cytoplasmic tail. Immunoprecipitation (IP; Flag) followed by 2D mass spectrometry revealed strong interactions of PP2A with cytoplasmic tail of CD25. Western blots showed additional strong interactions of the cytoplasmic tail of CD25 with inhibitory phosphatases PTEN, SHP1 and SHIP1. Importantly, reconstitution of myristoylated CD25 tail but not a mutant construct lacking the serine/threonine motif (S268A/T271A) rescued proliferation and survival defects of Il2ra-/- ALL cells.Conclusion: We identified CD25 as a surface receptor that mediates membrane recruitment of PP2A, PTEN, SHP1 and SHIP1, which balances fluctuations in signaling output from a pre-B cell receptor or its oncogenic mimic in ALL cells (e.g. BCR-ABL1 in Ph+ ALL). We propose that CD25-mediated negative feedback control stabilizes oncogenic tyrosine kinase signaling and mediates drug-resistance in Ph+ ALL and Ph-like ALL cells. Targeted inhibition using CD25-directed immunotoxins may be useful in new approaches to overcome drug-resistance in Ph+ ALL and Ph-like ALL. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1124: Transcriptional control of B cell identity restricts metabolic fitness in human leukemia

Abstract Oncogenic lesions in multi-potent progenitor cells often give rise to either B-cell or myeloid lineage leukemia. While transformed by the same oncogenes (e.g. BCR-ABL1, RAS), B-lineage and myeloid leukemias are distinct diseases. Given that oncogenic tyrosine kinase signaling (e.g. BCR-ABL1) imposes significant metabolic requirements on energy supply, biogenesis and metabolic fitness, we studied whether the divergent characteristics of myeloid and B-lineage leukemias have a metabolic basis. Metabolic analyses revealed that B-lineage acute lymphoblastic leukemia (Ph+ ALL) cells proliferate at maximum capacity of their glycolytic machinery. In contrast to myeloid leukemia (CML), B-lineage ALL cells lack metabolic adaptive fitness in response to metabolic fluctuations. C/EBPα-mediated reprogramming of B-lineage cells into the myeloid lineage induced glycolytic gene expression (Insr, Slc2a1, G6pdx, G6pd2, and Hk3). Frequent genetic lesions of transcription factors that determine B cell identity (IKZF1, PAX5, EBF1) partially mitigate B cell-instrinsic metabolic liability. Reconstitution of PAX5 expression in patient-derived B-lineage ALL cells reduced metabolic fitness by impacting glucose metabolism. Using genetic and metabolic experiments, we identified the metabolic liability observed in B-lineage ALL is in part dependent on the serine/threonine kinase LKB1. In agreement with previous studies, Cre-mediated deletion of Lkb1 induced proliferation in myeloid leukemia. Surprisingly, Lkb1 deletion led to apoptosis and decreased leukemogenic capacity in B-lineage leukemia. Consistent with the above observations, Arf, p53 and p27 levels were reduced in Lkb1-deficient myeloid leukemia cells, while Lkb1 deletion in B-lineage ALL cells up-regulated Arf, p53 and p27 levels. Enhanced glucose consumption and lactate production were observed in Lkb1-deficient myeloid leukemia cells. In contrast, loss of Lkb1 led to defective glycolytic and mitochondrial activity in B-lineage ALL. Lkb1 deletion in B-lineage ALL caused global accumulation of metabolites, suggesting that LKB1 is required for maintaining metabolic homeostasis. Moreover, loss of Lkb1 decreased protein levels of mitochondrial, anti-apoptotic BCL-2 family proteins, BCL-xL and MCL1, in B-lineage ALL. Reverse Phase Protein Array analyses revealed that LKB1 levels positively correlated with BCL-xL and MCL1 in patient-derived Ph+ ALL samples (n = 51) as well as other subtypes of B-lineage ALL (n = 183; MDACC, 1983-2007). Importantly, C/EBPα-mediated reprogramming of B-lineage ALL cells to the myeloid linage relieved dependency on LKB1. Taken together, we showed that transcriptional control of B cell identity causes unique metabolic liability. B-lineage ALL cells exhibit unique reliance on LKB1 for metabolic homeostasis and survival. Our findings revealed LKB1 as a potential therapeutic target in B-lineage ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Daniel Braas, Christian Hurtz, Seyedmehdi Shojaee, Huimin Geng, Valeria Cazzaniga, Carina Ng, Behzad Kharabi Masouleh, Yi Hua Qiu, Nianxiang Zhang, Kevin R. Coombes, Thomas Ernst, Giovanni Cazzaniga, Andreas Hochhaus, Steven Kornblau, Thomas Graeber. Transcriptional control of B cell identity restricts metabolic fitness in human 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 1124. doi:10.1158/1538-7445.AM2015-1124

Ikaros tumor suppressor function in pre-B ALL: potential role of Ikaros target gene Ctnnd1 (IRM10P.621)

Abstract Ikaros is a zinc finger transcription factor required for B-cell development and proper hematopoiesis, and an important tumor suppressor in developing lymphocytes. To understand the mechanism of Ikaros tumor suppressor function and potentially develop new and improved targeted therapies, it is important to elucidate the downstream target genes involved. With this aim, we combine mouse models of pre-B ALL and in vitro culture of human patient-derived pre-B ALL cells. A mouse model with targeted deletion of the fourth DNA-binding zinc finger of Ikaros resulted in loss of tumor suppressor function, with a limited set of deregulated genes useful to narrow down the list of putative relevant Ikaros target genes (Schjerven et al., 2013). To specifically address the role of Ikaros as a tumor suppressor in human pre-B ALL, we developed TET-regulated Ikaros expression in human pre-B ALL cells. This enables us to test specific Ikaros target genes from the mouse model, and allows for genome-wide expression analysis by RNA-seq to elucidate all genes downstream of Ikaros in human pre-B ALL cells. To help distinguish direct from indirect target genes, we have mapped the genome-wide binding sites of Ikaros in these human pre-B ALL cells by ChIP-Seq. This approach has identified the Ikaros target gene Ctnnd1. Ongoing experiments explore the potential role of Ctnnd1 and test the current hypothesis that Ikaros-mediated repression of Ctnnd1 limits CyclinD levels and leukemic growth in pre-B ALL.

IL2RA (CD25) Recruits Inhibitory Phosphatases to the Cell Membrane and Mediates Negative Feedback Control of STAT5 Signaling in Acute Lymphoblastic Leukemia

▪Background and hypothesis: CD25 (IL2RA, interleukin 2 receptor α chain) is a transmembrane protein with a 13aa cytoplasmic tail. CD25 cooperates with β- and γ-chains in binding IL-2, but does not contribute to cytokine signaling. During normal B cell development, CD25 is specifically upregulated on the surface of IL7-dependent pre-B cells and is also expressed on the surface of a subset of human pre-B ALL cases. CD25-expressing ALL is typically associated with poor clinical outcome. For these reasons, we studied the functional significance of CD25 expression on human pre-B ALL cells.Results: Flow cytometry and immunohistochemistry staining on a large panel of patient samples (n=416; MDACC, ECOG) revealed specific cell surface expression of CD25 in Ph+ ALL and Ph-like ALL, which are both high-risk subtypes of ALL. In agreement with selective expression on high-risk subsets, high expression levels of CD25 at the time of diagnosis were predictive of poor overall clinical outcome in these studies (P=0.005). BCR-ABL1 in Ph+ ALL and related tyrosine kinases in Ph-like ALL strongly activate STAT5, which then induces transcriptional activation of the IL2RA locus. Since Stat5 is also active during normal pre-B cell differentiation, we first analyzed B cell development in Il2ra-/- mouse bone marrow. Il2ra-/- B cell development was blocked at the pre-B cell stage, consistent with specific upregulation of CD25 on pre-B cells. In human Ph+ ALL cells, we found IL2RB and IL2RG were not co-expressed with CD25, suggesting a function of CD25 in Ph+ALL that is distinct from IL2 signaling. To test the biological significance of tyrosine kinase/STAT5-induced activation of CD25, we developed an Il2ra-/- mouse model for BCR-ABL1 pre-B ALL. Interestingly, the cytoplasmic tail of CD25 includes phosphorylation sites (S268 and T271) that are known substrates for serine/threonine phosphorylation by PKCα, which was reported to regulate protein phosphatase 2A (PP2A). To investigate interacting proteins with the cytoplasmic tail of CD25, we performed immune precipitation (IP) against the flag-tagged CD25-tail in primary Ph+ ALL cells which were transduced with either a CD25-tail-flag or an EV-flag vector. 2D mass spectrometry and Western blot on the IP products confirmed strong interactions with PKCα and PP2A. Weestern blot analysis confirmed additional interactions with inhibitory phosphatases including PTEN, PTPN6 (SHP1) and Inpp5d (SHIP1) in human Ph+ALL cells. In addition, both 2D MS and Westernblot showed recruitment of the Stat5-feedback inhibitors CISH, SOCS2 and SOCS3 at the CD25 cytoplasmic tail.Studying functional parameters of Il2ra-/-BCR-ABL1 ALL cells, we found impaired proliferation and colony formation capacity and drastically increased increased phosphorylation levels of pABLY412, pSTAT5Y694, pERKT202/Y204, pAKTS473, pP38T180/Y182 and p53. Reconstitution of CD25 expression restored normal phosphorylation levels of these molecules, as well as proliferation and colony formation.In a serial transplant setting, we observed that leukemia initiation in transplant recipients from Il2ra-/-BCR-ABL1 ALL cells required 10- to 100-times higher cell numbers, suggesting that CD25 contributes to leukemia initiation. In addition, CD25 expression is associated with a higher level of drug-resistance: In patient-derived pre-B ALL cells with mixed CD25Low and CD25High populations, the standard chemotherapy agent vincristine selectively induced apoptosis of in CD25Low but not CD25High ALL cells. An anti-CD25 immunotoxin drugs efficiently eradiated CD25High leukemia cells and thereby overcame drug-resistance against vincristine.Conclusions: Our studies identified CD25 as a surface receptor that mediates membrane recruitment of PP2A and CISH, SOCS2, negative feedback regulators of STAT5. CD25 is transcriptionally activated by STAT5 and therefore specifically expressed on high-risk ALL subtypes with oncogenic activation of the Stat5 pathway (Ph+ ALL and Ph-like ALL). We propose that CD25-mediated negative feedback control stabilizes oncogenic tyrosine kinase signaling and mediates drug-resistance in Ph+ ALL and Ph-like ALL cells. Targeted inhibition using CD25-directed immunotoxins may be useful in new approaches to overcome drug-resistance in Ph+ ALL and Ph-like ALL. DisclosuresNo relevant conflicts of interest to declare.

IFITM3 (CD225) Regulates CD19 Surface Expression and CD19-Mediated Activation of PI3K Signaling in Pre-B Acute Lymphoblastic Leukemia Cells

Background & Hypothesis:IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21 on mouse and human B cells. However, besides its interaction with CD19, specific functions of Ifitm3 in normal B cells development and, potentially leukemogenesis, are not known. Recent approaches based on CD19-specific chimeric antigen receptors (CAR) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (e.g. Grupp et al., N Engl J Med 2013). However, in some cases, CD19 CAR treatment was followed by ALL relapse developing from a clone that lacked CD19 surface expression. Therefore, we studied factors that can potentially regulate CD19 surface on normal and leukemic pre-B cells.Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in near-complete loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. Reconstitution of IFITM3 in patient-derived pre-B ALL cells rescued both CD19 expression and increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT, pointing to a central role of IFITM3 in regulating CD19/PI3K-AKT signaling in pre-B cells and pre-B ALL.Deletion of Ifitm3 also resulted in significant cell cycle arrest in the G0/G1 phase (P<0.001) and increased protein levels of p53 and p21 compared to wild-type pre-B ALL cells. Moreover, treatment with low dose adriamycin (25 ng ml-1) for induction of modest DNA damage significantly induced cellular senescence in Ifitm3-/- but not wild-type pre-B ALL cells. Consistent with Ifitm3-mediated proliferative defects of Ph+ ALL cells, Ifitm3-/- Ph+ ALL cells exhibited reduced self-renewal capacity (P=0.0004) decreased colony in colony forming assay compared to wild-type cells. Furthermore, Ifitm3 deficient B cell progenitors also showed significant inhibition of proliferation with inhibition of phosphorylation of Stat5Y694and c-Myc expression.4-hydroxytamoxifen (4-OHT)-inducible activation of CD19 rescued proliferative defects in Ifitm3 deficient B cell progenitors with release from G0/G1 cell cycle arrest associated with upregulation of phosphorylation of Stat5 Y694 and c-Myc expression. In addition, we found that CD19 positively regulates the surface expression of IL7R in B cell progenitors. Interestingly, CD19low population in Ifitm3 deficient B cell progenitors showed lower surface expression of IL7R compared to CD19high population. In contrast to B cell progenitors, forced expression of CD19 did not increase the proliferation of wild-type Ph+ ALL cells. Surprisingly, upregulation of CD19 induced apoptosis in Ifitm3-deficient Ph+ ALL cells with significant inhibition of BCR-ABL1 activity, phosphorylation of Stat5Y694and Bcl2 expression.Conclusion: These findings identify novel role of IFITM3 in regulating CD19 surface expression and CD19-Y513 mediated PI3K-AKT signaling in pre-B cells and pre-B ALL. Preliminary experiment showed that agonistic antibodies against IFITM3/CD225 increased CD19/PI3K-AKT signaling and proliferation in pre-B ALL cells and future studies will elucidate whether blocking antibodies (e.g. decoy Fc-fusion molecules) will have useful effects in targeting CD19/PI3K-AKT signaling in human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

BCL6 Enables RAS-Mediated Pre-B Cell Transformation in Childhood Acute Lymphoblastic Leukemia

Background & Hypothesis: The transcriptional repressor and proto-oncogene BCL6 has recently been identified as a therapeutic target in subtypes of diffuse large B cell lymphoma (DLBCL) and as mediator of a novel for of drug-resistance in Ph+ acute lymphoblastic leukemia (ALL; Duy et al., Nature 2011). A previous senescence rescue screen identified BCL6 as a key factor that bypassed senescence and thereby enabled RAS-mediated transformation of mouse embryonic fibroblasts (Shvarts et al., Genes Dev 2002). Since ~50% of pediatric cases of ALL carry genetic lesions that result in hyperactivation of the Ras-Erk pathway (Zhang et al., Blood 2012), we tested the role of Bcl6 in this large subgroup of childhood leukemia.Results: Mutations leading to hyperactivation of the Ras-Erk pathway are found in about 50% of childhood ALL cases (Zhang et al., 2012). Among 26 ALL xenografts, we found 9 cases with constitutive Erk-T202/Y204 phosphorylation, which was paralleled by elevated expression levels of BCL6 in these cases. Studying mouse pre-B cells that were engineered with a doxycycline-inducible NRASG12D mutant, we were able to directly measure the consequences of acute activation of the Ras-Erk pathway on BCL6 expression levels. First we incubated the cells with doxycycline to induce the expression of NRASG12D and then harvested the cells at different times point to test for BCL6 protein and mRNA expression levels. Interestingly, after 24h NRASG12D-TetO pre-B cells cells showed strong upregulation of BCL6 at the mRNA (352-fold) and protein level (15-fold). Upregulation of BCL6 in response to NRASG12D-activation was sensitive to treatment with the MEK kinase inhibitor PD325901, upstream of Erk, suggesting that BCL6 expression is a consequence of Erk activation in pre-B cells. Likewise, treatment of patient-derived pre-B ALL cells with the MEK inhibitor PD325901 reversed BCL6 expression, as demonstrated by quantitative RT-PCR and Western blot. From one patient, a diagnostic (KRAS wildtype) and a relapse sample with an acquired KRASG12V mutation were available. Consistent with specific expression of BCL6 in the KRASG12V relapse ALL sample, only KRASG12V ALL cells from the relapse but not wildtype cells from the diagnostic sample were sensitive to a retro inversoBCL6 peptide inhibitor (RI-BPI; Cerchietti et al., 2009).Development of a genetic mouse model for inducible ablation of Bcl6. These findings suggest an important role of BCL6 as a cofactor of RAS-driven pre-B cell transformation, comparable to previous findings in mouse embryonic fibroblasts. To directly test a mechanistic role of Bcl6 in RAS-mediated pre-B cell transformation, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Interestingly, Mb1-Cre x Bcl6fl/fl B cell lineage cells could be transduced with NRASG12D retroviral vectors, however these cells did not give rise to leukemia when injected into congenic recipients, whereas NRASG12D-transduced Bcl6fl/fl pro-B cells that retained Bcl6 function developed B cell lineage leukemia in all transplant recipients. In a second experiment, we transformed Bcl6fl/fl pro-B cells with NRASG12D and induced Cre with a second, tamoxifen-inducible vector in full-blown leukemia cells. Acute ablation of Bcl6 in NRASG12D ALL cells completely abrogated the ability of NRASG12DALL cells to form colonies in methylcellulose and resulted in rapid apoptosis and depletion from the cell culture. We conclude that BCL6 is not only required for the initiation of RAS-driven ALL in vivo but also for the maintenance of fully established RAS-driven leukemia.Conclusion: These findings provide genetic evidence for BCL6 function as a critical cofactor of RAS-mediated transformation in childhood ALL. Inhibition of BCL6 in RAS-driven ALL may be useful to prevent leukemia relapse after initial remission (Bcl6-dependent leukemia-initiation) and also to achieve profound remission by combining conventional cytotoxic therapies with BCL6 inhibition (e.g. RI-BPI). DisclosuresNo relevant conflicts of interest to declare.

Effector-mediated eradication of precursor B acute lymphoblastic leukemia with a novel Fc-engineered monoclonal antibody targeting the BAFF-R.

B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia (pre-B ALL) cells, but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here, we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild-type cells, but were more sensitive to drug treatment in vitro. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti-BAFF-R monoclonal antibody optimalized for FcRγIII-mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated natural killer cell-mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti-pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted.

Identification of Sphingosine Kinases As Therapeutic Targets in B-Lineage Acute Lymphoblastic Leukemia

AbstractAbstract 1499Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and survival. S1P is produced by the sphingosine kinases, SphK1 and SphK2. SphK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SphK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SphK1. Little is known about the interaction of SphKs with intracellular signalling pathways. Here we assess the relevance of SphKs in B-lineage acute lymphoblastic leukemia (B-ALL).Gene expression signatures indicative of activation of SphK1 or SphK2 were not publicly available. Therefore we treated ALL cell lines with the SphK1 or SphK2 specific inhibitors SK1-I and ABC294640 respectively, and analysed gene expression by microarray. Although a signature for SphK1 was not obtained, two independent methods of analysis generated SphK2 gene signatures that segregated control and drug treated cell lines. The signatures consisted of 11 and 35 genes and included reduced expression of the NF-κB inhibitor TRIB3 (NF-κB activity is commonly up regulated in ALL), the C/EBP family transcription factor DDIT3, the protein phosphatase 1 regulatory subunit PPP1R15A and the sphingolipid/cholesterol transporter ABCA1, all validated by quantitative RT-PCR. These gene signatures were used to interrogate a large publicly available gene expression dataset (GSE28497) obtained from pediatric ALL patients at the time of diagnosis. SphK2 activity signatures were more highly expressed in ALL samples (p=0.001 and p=0.027 for the smaller and larger signatures respectively) than normal B-cell progenitors. Although SPHK1 or SPHK2 genes were not over expressed in this dataset, SphK1 protein levels were increased in ALL cell lines and in patient samples.The importance of SphK1 and SphK2 in the development of ALL was examined by transducing B-cell progenitors isolated from WT, SphK1−/− or SphK2−/− mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated wild type C57BL/6 mice. Twenty-two of 29 mice receiving cells from WT animals developed ALL, with a mean survival of 52.6 days (95% CI 42.3–62.8 days). Mice receiving cells lacking Sphk1 or Sphk2 had a significantly reduced incidence of ALL development with 14 of 30 (mean survival 71.6 days, 95% CI 60.5–82.8 days) and 16 of 29 animals (mean survival of 68.7 days, 95% CI 57.9–79.5 days) respectively (p=0.001). Lymphoblasts with a B-cell progenitor phenotype (B220+, CD19+, IgM−, CD11b−) were present in blood films and livers from leukemic mice and cells recovered from these animals produced a rapidly fatal ALL in secondary recipients. The presence of BCR/ABL and the expected deletion of Sphk1 or Sphk2 were confirmed by PCR in all murine leukemias examined.We have previously reported that inhibitors of SphK1 and/or SphK2 inhibit proliferation and induce cell death in ALL cell lines, and that these agents can synergize with imatinib in Ph+ ALL cell lines. We have furthered these studies to demonstrate that patient derived ALL cells similarly respond to SphK1 and SphK2 inhibition in vitro. The SphK2 inhibitor ABC294640 (100mg/kg) reduced plasma S1P levels in mice consistent with our previous reports of reduced ALL burden following ABC294640 treatment, and using a NOD/SCID γc−/− xenograft model of human ALL we have now shown that ABC294640 extends survival of ALL-bearing mice (p=0.0012 for Ph− xenograft) and further extends the survival when combined with imatinib treatment in mice engrafted with a human Ph+ ALL patient sample (p=0.044).This is the first report to suggest that sphingosine kinase 2 activity is increased in ALL, providing support for targeting SphK2 as a therapeutic strategy. Loss of SphKs reduces the incidence of ALL in a murine model of BCR/ABL-driven disease and ABC294640 reduces disease and extends survival in a human xenograft model of ALL. This compound synergizes with a number of potential therapeutic agents and further extends survival in a xenograft model of Ph+disease when combined with imatinib. This has potential to translate into a useful anti-leukemic strategy. Disclosures:No relevant conflicts of interest to declare.

585 NOXA as mediator for drug-induced apoptosis - molecular studies in patient-derived ALL cells

585 NOXA as mediator for drug-induced apoptosis - molecular studies in patient-derived ALL cells

Role of Survivin in Drug Resistant B-Cell Acute Lymphoblastic Leukemia.

Abstract 10Survivin/BIRC5, an inhibitor of apoptosis (IAP) protein,is critical for the survival and proliferation of cancerous cells and has become the target of an increasing number of preclinical novel therapies against primarily solid tumors. Survivin is expressed in AML and ALL cells and has been implicted in leukemia relapse. Here we test the hypothesis that Survivin is critical to chemotherapeutic drug resistance in ALL. To test this hypothesis, we initially compared survivin mRNA levels in both patient-derived B-ALL cells and B-ALL cell lines versus normal B-cells in various stages of development using real-time PCR. ALL cells encompassing various cytogenetic subgroups showed significantly greater mRNA levels of Survivin versus normal B cell precursors ranging from 2 to greater than 20-fold versus controls. To determine whether Survivin contributes to drug resistance, we lentivirally overexpressed Survivin in primary B-ALL and B-ALL cell lines. Overexpression of Survivin attenuated the effect of Vincristine on ALL cell proliferation when compared to ALL cells transduced with empty vector controls. Vincristine IC50 value determination for the control was 0.1 nM, whereas Survivin overexpression resulted an IC50 value of 10 nM (p<0.01). Similarly, significantly higher concentrations of L-Asparaginase (>0.01 U/l vs ab >0.1; p<0.05) and Dexamethasone (0.1 nmol/l vs >10 nmol/l; p<0.013) were needed to achieve drug cytotoxicity. We conclude that Survivin overexpression decreases sensitivity of ALL cells to standard chemotherapy. Conversely, lentivirally-mediated survivin shRNA knockdown of the same cell type significantly sensitized a greater percentage of the leukemia to 10nM vincristine versus non-silencing controls. Survivin shRNA targeting resulted in a 30.2% greater affected population than controls, assayed using MTT (p<0.003). Overexpression or knockdown of Survivin was confirmed by Western Blot and real-time PCR. Of note, expression levels of Survivin is heterogeneous within the same ALL sample: Transduction of patient-derived ALL cells with a Survivin-GFP reporter construct revealed that a small subpopulation of Survivin-GFP high ALL exhibiting approximately 7-fold higher levels of endogenous Survivin than bulk leukemia cells. Consistent with cell cycle-dependent regulation of Survivin, Survivin-GFP high ALL were enriched in G2/M phase of the cell cycle. In addition, endogenous variations of Survivin protein levels were in the same range as changes in Survivin protein levels in overexpression and knockdown experiments, which indicates that these experiments tested physiologically occurring levels of Survivin. Next, we determined whether Survivin contributes to self-renewal of drug resistant ALL cells. Survivin overexpression of primary samples yielded three times more colonies, than controls in a primary plating CFU assay (p<0.019). Taken together, our data suggests that targeting Survivin in B-ALL may sensitize to chemotherapy and highlights the role of Survivin in drug resistant B-ALL as a target for novel therapies. Disclosures:No relevant conflicts of interest to declare.

XmAb5574: In vitro and in vivo efficacy of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma and leukemia and its effect on B cells in non-human primates

3078 Background: CD19 is a pan-B cell surface receptor expressed from early stages of pre-B cell development through terminal differentiation into plasma cells. It is an attractive target for cancers of lymphoid origin since it is expressed on the vast majority of Non- Hodgkin Lymphoma (NHL) and leukemia cells. The majority of NHL patients relapse after the current treatment regimen of chemotherapy combined with rituximab (CHOP-R), thus salvage regimens with non-cross resistant antibody therapies are warranted. We present the characterization of a novel humanized Fc-engineered anti-CD19 antibody (XmAb5574). Methods: We applied XmAb technology to a chimeric CD19 antibody to humanize it, increase its affinity for Fc gamma receptors, improve its effector function, and thus significantly increase its antitumor potency. This approach yielded a pre-clinical candidate (XmAb5574) that was tested in a series of in vitro cytotoxicity assays and in mouse xenograft models of NHL. We also tested the depletion of B cells in peripheral blood, lymph nodes, bone marrow and spleen of cynomolgus monkeys. Results: XmAb5574 is a novel anti-CD19 antibody that is highly cytotoxic against a panel of lymphoma and leukemia cell lines. This antibody does not appreciably internalize, and in vitro it mediates ADCC (EC50 ∼ 1 - 0.03 ng/ml) and phagocytosis (EC50 ∼ 3 - 0.1 ng/ml) superior to that of rituximab and alemtuzumab. XmAb5574-mediated ADCC is also significantly increased relative to rituximab in patient-derived ALL, CLL and MCL cells. XmAb5574 exhibits in vitro apoptotic activity that is significantly more potent than that of rituximab. Xenograft mouse models, Ramos and Raji sc and iv, show that XmAb5574 has robust protection in both protocols. Finally, we observed sustained depletion of peripheral and lymphoid B cells in cynomolgus monkeys dosed with XmAb5574. Conclusions: The main features of XmAb5574 are: i) improved in vitro cytotoxicity in tumor cells, ii) robust antitumor activity in mouse xenograft models, and iii) B cell depletion in cynomolgus monkeys. Thus XmAb5574, our anti-CD19 Fc variant antibody engineered for increased effector function, is a promising next-generation immunotherapeutic for leukemias and lymphomas. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Xencor Xencor

XmAb™ Fc Engineered Anti-CD19 Monoclonal Antibody with Enhanced In Vitro Efficacy Against Lymphoma and Leukemia.

CD19 is a pan-B cell surface receptor expressed from early stages of pre-B cell development through terminal differentiation into plasma cells. It is an attractive target for cancers of lymphoid origin since it is expressed on the vast majority of Non-Hodgkin Lymphoma (NHL) as well as different types of leukemia, including those (e.g. pediatric ALL) lacking CD20 expresion. The majority of NHL patients will relapse after the current treatment regimen of chemotherapy combined with rituximab (CHOP-R) despite major improvements in response rates and progression free survival. Thus salvage regimens with novel non-cross resistant antibody therapies are warranted. Here we present the characterization of a novel Fc-engineered and humanized anti-CD19 antibody (XmAb™ CD19) that was generated using our XmAb™ antibody engineering technologies. This novel antibody is highly cytotoxic against a panel of lymphoma and leukemia cell lines as well as primary cancer cells. The main features of this antibody are:i.increased affinity for Fc gamma receptors (FcgR),ii.improved effector function, andiii.significantly increased antitumor potency.Humanization and affinity maturation technologies were applied to this antibody in order to:i.decrease immunogenicity,ii.increase affinity, andiii.increase stability of the engineered antibody.Since internalization is expected to impact a naked antibody's effector functions, we assayed its internalization rate using Eu-labeled XmAbCD19 and observed an unexpectedly low rate of internalization. Therefore, we proceeded to investigate several direct and indirect (Fc-mediated) mechanisms of antibody-mediated cytotoxicity. The potency of XmAbCD19 in antibody-dependent cell-mediated cytotoxicity (ADCC) increased 10- to 100-fold relative to a native/non Fc-engineered version (CD19-IgG1) of the antibody in a screen of 16 NHL and leukemia cell lines (chronic lymphocytic leukemia [CLL], B-cell acute lymphoblastic leukemia [B-ALL], hairy cell leukemia [HCL], follicular lymphoma [FL], mantle cell lymphoma [MCL], chronic myelogenous leukemia [CML], and Burkitt's lymphoma [BL]). ADCC potency (EC50) and efficacy (% Lysis) of the Fc-engineered anti-CD19 antibody were superior to that of rituximab: in CLL - 10- and 1.5-fold higher, in B-ALL - 10- and 100-fold higher, and in HCL - 6- and 1.2-fold higher, respectively; unlike XmAbCD19 native CD19-IgG1 mediated little ADCC. Moreover, XmAbCD19 mediated potent ADCC of primary patient-derived ALL cells that was also significantly increased in potency and efficacy relative to rituximab and CD19 IgG1. Furthermore, XmAbCD19 demonstrated 50-fold increased antibody-dependent cellular phagocytosis (ADCP) relative to CD19-IgG1. Finally, XmAbCD19 exhibited robust anti-proliferative activity that was 10-fold more potent than that of rituximab. In order to facilitate preclinical toxicology studies with XmAbCD19, we assayed its cross-reactivity with non-human primate (NHP) species CD19 (cynomolgus and rhesus monkeys). In contrast to the parental murine antibody, the affinity-optimized humanized XmAbCD19 reacted with B cells from both NHP species. In summary, our data suggest that our anti-CD19 Fc variant antibody engineered for increased effector function is a promising next-generation immunotherapeutic for a variety of leukemias and lymphomas.