Patient-derived pre-B Acute Lymphoblastic Leukemia Research Articles

Abstract 93: Transcriptional control of glucocorticoid responses in leukemia

Abstract Glucocorticoids (GCs) are central to all major therapy regimens for pre-B cell-derived acute lymphoblastic leukemia (ALL), but have no activity in myeloid leukemia. Such divergent responses represent an empirically established clinical standard; however, neither the mechanism by which GCs induce cell death nor the biological basis for the distinct responses in B-cell and myeloid leukemias is clear. Studying patient-derived samples revealed that NR3C1 (glucocorticoid receptor) levels were 6- to 20-fold higher in pre-B ALL compared to chronic myeloid leukemia (CML). High levels of Nr3c1 were reduced upon B- to myeloid-lineage conversion, suggesting that regulation of NR3C1 expression and GC responsiveness depend on a B-cell transcriptional program. B-cell transcription factors (e.g. PAX5, IKZF1) are critical for B-cell development, yet they are genetically lesioned in more than 80% of pre-B ALL cases. Despite such high frequency, the significance of these inactivating lesions remains elusive. Combining ChIP-seq and RNA-seq analyses, we identified a novel B-cell transcriptional program for activation of NR3C1 and its transcriptional target TXNIP (a negative regulator of glucose uptake). Reconstitution of PAX5 or IKZF1 expression in haploinsufficient patient-derived pre-B ALL cells increased NR3C1 and TXNIP levels. Conversely, expression of dominant negative mutant of PAX5 or IKZF1 abolished NR3C1 expression. Loss of Nr3c1 or Txnip in murine BCR-ABL1-driven pre-B ALL cells resulted in survival advantage in competitive growth assays. Importantly, loss of Nr3c1 or Txnip significantly elevated glucose uptake, lactate production and cellular ATP levels. These findings suggest that GCs induce cell death by exacerbating glucose and energy depletion. Notably, reconstitution of PAX5 or IKZF1 rendered haploinsufficient patient-derived pre-B ALL cells more sensitive to dexamethasone (dex) treatment. In contrast, dominant-negative PAX5 or IKZF1 largely de-sensitized pre-B ALL cells expressing wildtype PAX5 or IKZF1. These findings suggest that B-cell transcription factors set the threshold for GC responsiveness in pre-B ALL. Since relapsed ALL cells often acquire GC resistance, drug-combinations may be useful to prevent GC-resistance. As expected, loss of Nr3c1 abrogated responses to GCs. Interestingly, loss of Txnip also largely rescued GC-induced cell death in pre-B ALL cells. On this basis, we tested drug interactions between GCs and TXNIP agonists, 3-O-methylglucose (3-OMG) and D-allose. Treating patient-derived GC-refractory pre-B ALL cells with 3-OMG or D-allose strongly synergized with GC-treatment. Collectively, our findings provide a mechanistic explanation for the empiric finding that GCs are effective in the treatment of B-cell but not myeloid malignancies, and identify TXNIP as a novel therapeutic target in pre-B ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Zhengshan Chen, Gang Xiao, Jae Woong Lee, Kadriye Nehir Cosgun, Huimin Geng, Valeria Cazzaniga, Hilde Schjerven, Ross A. Dickins, Markus Muschen. Transcriptional control of glucocorticoid responses in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 93. doi:10.1158/1538-7445.AM2017-93

Erk Negative Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation ofoncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation ofErk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels,we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse modelsfor pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance.

Erk and Stat5 Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

▪Background and Hypothesis: Targeted therapy of cancer typically focuses on the development of agents that will inactivate a transforming oncogene. In this study, we tested the concept that besides the oncogene itself, factors that enable permissiveness of a normal cell to oncogenic signaling represent a novel class of therapeutic targets. This hypothesis was based on three findings. First, acute activation of oncogenes in normal pre-B cells typically caused immediate cell death, unless pre-B cells were capable of adapting quickly enough to a high level of signaling output. Second, few surviving pre-B cell clones achieved permissiveness to oncogenic signaling by strong activation of negative feedback control of Erk and Stat5. Third, robust feedback control of Erk and Stat5 distinguishes normal pre-B cells from fully transformed pre-B acute lymphoblastic leukemia (ALL) cells.Results: To test the significance of strong feedback control of Erk and Stat5 signaling in pre-B ALL cells, we developed genetic loss-of function models for six central molecules in Erk (DUSP6, SPRY2, ETV5) and Stat5 (Cish, SOCS2, SOCS3) feedback control. Genetic deletion of the sprouty family Ras inhibitor Spry2, the Erk dual specificity phosphatase Dusp6 and their transcriptional activator Etv5, decreased robustness of Erk feedback control and compromised oncogenic transformation in mouse models for pre-B ALL. Likewise, ablation of Stat5 feedback control through deletion of the suppressors of cytokine signaling (SOCS) family molecules Cish, Socs2 and Socs3 reversed permissiveness of pre-B cells. Studying deletion of Spry2 (Erk) and Cish (Stat5) in an in vivo transplant model using inducible, Cre-mediated deletion of Spry2 and Cish in pre-B ALL cells confirmed that Erk and Stat5 feedback control are essential for malignant transformation and development of lethal leukemia. Genetic deletion of Erk (Dusp6, Spry2, Etv5) and Stat5 (Cish, Socs2, Socs3) feedback control impairs leukemic transformation of pre-B cells.Searching for factors that restrict permissiveness to oncogene signaling, we identified the pre-B cell tumor suppressor IKZF1, which is deleted in a large fraction of pre-B ALL cases. IKZF1 directly bound to and transcriptionally repressed multiple promoters of Erk and Stat5 feedback control and IKZF1 deletion raised the limit of maximum allowable oncogene signaling strength in pre-B ALL cells. We propose that the pre-B cell tumor suppressor IKZF1 functions as transcriptional repressor of Erk and Stat5 feedback control and thereby retains pre-B cells in a Non-permissive state.Clinical relevance: To assess potential usefulness of this finding for the development of future treatment strategies, we tested the effect of a specific small molecule inhibitor of DUSP6, E-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), which was designed as an allosteric inhibitor of the interaction between DUSP6 and phospho-ERK1/2. Interestingly, BCI acutely subverted Erk feedback control and selectively induced cell death in pre-B ALL cells. Small molecule inhibition of DUSP6 was sufficient to overcome conventional mechanisms of drug-resistance in pre-B ALL and selectively killed patient-derived pre-B ALL cells in a leukemia transplant model. BCI treatment, similar to Dusp6-deletion in our leukemia mouse model, led to the accumulation of P53 and ARF in patient-derived pre-B ALL cells. In addition, small molecule inhibition of DUSP6 had strong selective activity on drug-resistant patient-derived pre-B ALL cells that were injected into NOD/SCID transplant recipient mice. These findings identify permissive negative feedback control of oncogenic signaling as a previously unrecognized vulnerability of pre-B ALL cells and a new class of potential therapeutic targets.Conclusion: Targeting negative feedback regulation of both Erk and Stat5 signaling for the treatment of pre-B ALL seems counter-intuitive because it represents effectively the opposite of current efforts of targeted inhibition of oncogenic signaling. Our results, however, demonstrate that a robust negative feedback regulation is required for the leukemic transformation and development of fatal leukemia in pre-B ALL. We demonstrate that feedback control of Erk and Stat5 signaling represents a previously unrecognized vulnerability and, potentially, a novel class of therapeutic targets in human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Lineage-Specific Metabolic Reprogramming Reveals LKB1 as Therapeutic Target in Acute Lymphoblastic Leukemia

Background & Hypothesis: The Philadelphia chromosome encoding the oncogenic BCR-ABL1 (Ph+) tyrosine kinase represents a common transforming oncogene in both chronic myeloid leukemia (CML) and a subset of pre-B acute lymphoblastic leukemia (ALL). While both diseases are driven by the same oncogene, biological and clinical characteristics differ between CML and Ph+ ALL. Given that oncogenic signaling from potent tyrosine kinases like BCR-ABL1 imposes significant metabolic requirements on energy supply, biogenesis and ability to survive metabolic stress, we investigated whether the divergent characteristics of BCR-ABL1 CML and Ph+ ALL have a metabolic basis.Results: Interestingly, metabolic analyses demonstrated that patient-derived CML chronic phase (CML-CP) and Ph+ ALL cells have distinguishable glycolytic profiles. Consistent with a higher proliferative rate, Ph+ ALL cells rapidly exhausted the capacity of their glycolytic machinery when compared to CML-CP cells. Furthermore, when comparing their glycolytic reserve (i.e. spare glycolytic capacity needed to compensate for loss of mitochondrial ATP production), Ph+ ALL cells were shown to have a lower glycolytic reserve compared to CML-CP cells. These findings suggest that Ph+ ALL cells have less flexibility in adapting to metabolic stress. Searching for factors that mediate metabolic reprogramming to support rapid cellular proliferation and promote adaptation to metabolic stress in a lineage-specific manner, we identified the tumor suppressor LKB1 and its substrate AMPK which coordinate metabolism with cell growth.To elucidate the potential lineage-specific functions of LKB1, genetic mouse models for 4-hydroxytamoxifen (4-OHT)-inducible deletion of Lkb1 in BCR-ABL1-driven myeloid leukemia (CML-like) and pre-B ALL (Ph+ ALL-like) were developed. Cre-mediated deletion of Lkb1 in myeloid leukemia mediated proliferation and stimulated aerobic glycolysis, as reflected by increases in both glucose consumption and lactate production. These findings are in agreement with previous findings in solid tumors and are consistent with the role of LKB1 as a tumor suppressor. In striking contrast, deletion of Lkb1 in pre-B ALL cells induced apoptosis and cell cycle arrest. In vivo, Lkb1 deletion in pre-B ALL cells delayed leukemia initiation and prolonged overall survival of transplant recipient mice. Furthermore, bioenergetics measurements of glycolytic and mitochondrial functions in response to various conditions of metabolic stress revealed that loss of Lkb1 function in pre-B ALL resulted in impaired metabolic adaptation. Likewise, metabolite profiling revealed that deletion of Lkb1 in pre-B ALL cells resulted in altered levels of metabolites from glycolysis, the TCA cycle, the pentose phosphate pathway, nucleotide metabolism, currency metabolites, amino acid metabolism and fatty acid metabolism. Importantly, C/EBPα-mediated lineage reprogramming of pre-B ALL cells into the myeloid lineage alleviated their dependency on Lkb1.Similar to observations made with Cre-mediated deletion of Lkb1 in a model for BCR-ABL1 pre-B ALL, small molecule inhibition of AMPK using BML-275 (an ATP-competitive inhibitor) induced apoptosis in patient-derived pre-B ALL cells. Moreover, BML-275 had deleterious effects on metabolic adaptation in patient-derived pre-B ALL cells, but not in CML-CP cells. Finally, BML-275 synergized with pharmacological inhibition of PI3K/Akt signaling in inhibiting glycolytic function and eradicating patient-derived pre-B ALL cells. Combining BML-275 and a PI3K inhibitor (BKM120) exerted significantly more potent inhibitory effect on pre-B ALL progression than each agent alone, prolonging the overall survival of recipient mice.Conclusions: Taken together, our findings demonstrate that LKB1/AMPK signaling plays lineage-specific roles in BCR-ABL1-driven CML and pre-B ALL. While LKB1/AMPK is an established tumor suppressor pathway in multiple cell types, our findings reveal a unique and previously unrecognized vulnerability of pre-B ALL cells. DisclosuresNo relevant conflicts of interest to declare.

FOXM1 Mediates Drug-Resistance and Represents a Therapeutic Target in Pre-B Acute Lymphoblastic Leukemia

Background & Significance: Pre-B acute lymphoblastic leukemia (ALL) emerges in virtually all cases from B cell precursors that are arrested at the pre-B cell receptor checkpoint. In a gene expression survey of early B cell development, we found specific upregulation of FOXM1 at this developmental stage. FOXM1 belongs to the forkhead box transcription factor family and is a key regulator of cell growth by promoting cell cycle progression and has been implicated in progression of solid tumors. Therefore, we characterized the function and regulation of FOXM1 in normal B cell development as well as in pre-B ALL.Results: First, we verified the upregulation of FOXM1 during B cell development by qRT-PCR on sorted human and murine B cell progenitor populations. Then, we crossed Mb1-Cre tg mice to a Foxm1 conditional knockout mouse model (Foxm1fl/fl) and analyzed the early B cell populations according to the Hardy fractions. Despite the observed high expression of Foxm1 mRNA in fraction C’ and D, Foxm1 deletion did not alter B cell development. In order to investigate a potential role of FOXM1 in transformed B cells, we compared FOXM1 protein levels in patient-derived pre-B ALL samples with healthy B cells and B cell precursors and found 10-60-fold higher expression in the transformed B cell progenitors. To evaluate a potential predictive value of FOXM1 levels in patient-derived ALL samples, we measured FOXM1 mRNA levels at the time of diagnosis which strikingly correlate with risk stratification of ALL (intermediate-risk ALL n=31 vs. high risk ALL n=21; P=7.3e-5; BFM-REZ 2002). To further study the function and regulation of FOXM1, we cultured murine B cell precursors in the presence of IL7 and induced transformation with a retroviral BCR-ABL1 expression vector. BCR-ABL1 expression increased levels of FOXM1 compared to the normal IL7-dependent pre-B cells. Short-term inhibition of BCR-ABL1 did not affected protein levels of FOXM1. However, after 4 days of tyrosine kinase inhibition (TKI) treatment, FOXM1 protein levels were significantly downregulated in a dose-dependent manner. BCL2 overexpression prevented apoptosis induction by TKI but FOXM1 downregulation was retained. In addition, we found evidence that inactivation of FOXO factors by the PI3K/AKT pathway contributes to high FOXM1 expression in Ph+ ALL. Overexpression of a constitutively active form of Akt to prevent activation of FOXO factors in the presence of TKI abrogated FOXM1 downregulation. Similarly, BCR-ABL1+ ALL derived from FOXO3a knockout mice prevented TKI-mediated FOXM1 reduction. Overexpression of a constitutively active form of FOXO3a but not FOXO1 significantly reduces levels of FOXM1 expression. In line with this, we found a significant inverse correlation of FOXM1 with FOXO3A mRNA levels in Ph+ ALL patients from the ECOG E2993 trial. However, the requirement of long-term treatment indicates, that, in addition to FOXO3a activation, epigenetic regulation of the FOXM1 promoter downstream of BCR-ABL1 is required. Consistent with this finding, the FOXM1 promoter region was found to be de-methylated in a large fraction of ALL. In order to further study FOXM1 function, we transduced pre-B cells derived from Foxm1fl/fl mice with BCR-ABL1 and with an inducible ERT2-Cre vector. Deletion of Foxm1 in BCR-ABL1-driven leukemia decreases cell viability, colony formation, and proliferative capacity in vitro as well as leukemia formation in vivo. FOXM1-deleted ALL cells revealed a strikingly higher sensitivity towards TKI-treatment compared to the control cells in Imatinib dose-response curves (IC50 EV:420 nM vs IC50 Cre-ERT2: 160 nM) as well as annexin V staining. We identified the ROS scavenger Catalase as a critical target of FOXM1 in mediating this drug resistance. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide and the natural occurring antibiotic Thiostrepton. Both bind FOXM1 and inhibit its function as shown by reduced mRNA expression of FOXM1 target genes (Cyclin B1, PLK1, AURKB) and induced apoptosis in ALL and prolonged survival of patient-derived ALL transplant recipient mice.Conclusion: We have identified a critical function of the transcription factor FOXM1 in mediating proliferation and drug-resistance in B cell lineage ALL, but not in normal B cell progenitors and validated FOXM1 as a therapeutic target in a large fraction of drug-resistant B cell lineage ALL. DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of Clonal Evolution of Pre-Leukemic Clones in Childhood Pre-B Acute Lymphoblastic Leukemia

Background and hypothesis: Childhood pre-B acute lymphoblastic leukemia (ALL) can frequently be retraced to a pre-leukemic clone carrying a prenatally acquired genetic lesion (e.g. ETV6-RUNX1gene rearrangement). After birth, pre-leukemic clones can acquire secondary mutations and, hence, evolve towards overt leukemia. While this concept is well established, the mechanism(s) driving clonal evolution are not known. Epidemiological findings hint to a role of delayed childhood infections and chronic inflammation as etiologic factors of childhood ALL, but do not illuminate mechanism of clonal evolution of pre-leukemic cells. In this study, we demonstrate that cooperation between the AID cytosine deaminase and the RAG1/RAG2 V(D)J recombinase promotes acquisition of secondary genetic lesions that promote progress of pre-leukemic B cell precursors towards full-blown leukemia.Results: The enzymatic activity of RAG1/RAG2 (VDJ recombination) and AID (somatic hypermutation, class-switch recombination) are strictly segregated to early and late stages of B cell development, respectively. While RAG1 and RAG2 are actively expressed at stages of early B cell development (bone marrow and fetal liver) that give rise to pre-B ALL, little is known about the function of AID in early B-lymphopoesis. As the involvement of AID in pre-B leukemic clonal evolution is incumbent on its expression during early stages of B-lymphopoesis, we tested CD19+ pre-B cells isolated from human bone marrow (BM) for indicators of AID activity, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Interestingly, most pre-B cell clones carry rearranged Ig VH region genes that are mutated at low levels (average mutation frequency 26 x 10-3 bp). Likewise, pre-B cells isolated from fetal liver tissues (three donors; 10-19 weeks of gestation) carried Ig VH region genes mutated at low levels (average mutation frequency 14 x 10-3 bp). In addition, about one third of fetal liver pre-B cells had undergone CSR to Cγ3, Cγ1 and Cα regions. These findings highlight the previously unknown function of AID in two important sites of early human B-lymphopoesis. Based on these results, we hypothesized that a specific B cell subset during early pro- and pre-B cell differentiation can concomitantly express both AID and the RAGs and, hence, would be particularly susceptible to clonal evolution of cells that carry a pre-leukemic lesion.Our subsequent studies identified late pre-B cells (Fraction D) as a natural subset of increased genetic vulnerability. Late pre-B cells downregulate IL7 receptor/Stat5 signaling, which enables expression of RAG1 and RAG2 and immunoglobulin light chain gene rearrangement. Loss of IL7 receptor/Stat5 signaling also removes an important safeguard against premature expression of AID. Therefore, late pre-B cells are poised to express AID at high levels in response to inflammatory stimuli (e.g. LPS) in concurrence with RAG1 and RAG2. Studying clonal evolution of patient-derived pre-B ALL cells, we found evidence for concomitant AID and RAG1/RAG2 activity. Further studying a genetic mouse model for pre-leukemic pre-B cells carrying ETV6-RUNX1, we found that repeated exposure to LPS can cause overt leukemia but not in the absence of either AID or Rag1. Additionally, whole exome sequencing of human B cell clones that were engineered to express AID, RAG1/RAG2 alone or in combination revealed that concurrent expression of AID with RAG1/RAG2 dramatically increased the frequency of structural chromosomal lesions.Conclusion: Consistent with epidemiological findings on the etiology of childhood ALL, we conclude that reduced cytokine signaling (here, IL7R) in late pre-B cells renders pre-leukemic clones distinctively vulnerable to genetic lesions that can be acquired in the context of repeated exposure to inflammatory stimuli (e.g. chronic and recurrent infections during childhood). Our results support a role for AID and RAGs cooperation for the generation of secondary lesions in leukemia subgroups that require additional leukemogenic events, and therefore, provide the genetic and molecular basis to support the Delayed Infections Hypothesis for leukemia progression in children. DisclosuresNo relevant conflicts of interest to declare.

PI3Kdelta Inhibitor, CAL-101, De-Adheres Primary Pre-B ALL from VCAM-1 and Induces Apoptosis in Primary Pre-B ALL

BACKGROUND: Relapse remains a major problem in the treatment pre-B acute lymphoblastic leukemia (ALL). 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 (CAM-DR) 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. However, it remains elusive how this target can be therapeutically harnessed. Therefore, we investigated if directly targeting the PI3K/AKT pathway downstream of α4 can overcome CAM-DR in pre-B ALL using a novel, FDA-approved PI3Kδ inhibitor, CAL-101METHODS: For in vitro studies, murine integrin α4 conditional knockout mouse model (α4fl/fl) oncogenically transformed with BCR-ABL1+ and patient-derived (primary) pre-B ALL cells (Philadelphia chromosome negative) co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells were used. Annexin V/7-AAD staining was used for viability determination by flow cytometry and Western Blot was used for determination of PI3Kδ and p-Akt expression. For in vivo experiments, we used a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL.RESULTS: Integrin α4fl/fl cells were cultured under lymphoid-skewing conditions and oncogenically transformed using BCR-ABL1 (p210). Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which α4 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen while plated on murine VCAM-1. In the absence of a4, mouse pre-B ALL cells contain markedly reduced levels of pAKT (ser473). In a parallel experiment, we observed in primary pre-B ALL cells co-cultured with OP9 cells an increase in p-AKT compared to media-only cultured ALL cells indicating activation of the PI3K/AKT pathway upon adhesion to stroma. Under starvation conditions, primary pre-B ALL cells (LAX7R) were incubated for 4 hours without serum or stroma, and subsequently treated for 48 hours with an integrin α4 blocking antibody or with control IgG4. By Western Blot, we detected decreased levels of activated p-AKT in the cells with α4 blockade, but not with control, indicating that the AKT pathway is affected by modulation of α4.To directly target the PI3K/AKT pathway, we used CAL-101, a small molecule inhibitor highly selective for the δ isoform, which was recently FDA-approved for treatment of CLL and Non-Hodgkin Lymphoma (NHL). Previously, in primary samples from CLL patients and ALL cell lines, CAL-101 blocked PI3Kδ-AKT signaling and promoted apoptosis. However, a preclinical evaluation in primary pre-B ALL patients in vitro and in vivo are missing. For these reasons, we used CAL-101 in the following experiments as a specific PI3Kδ inhibitor in primary pre-B ALL. Expression of PI3Kδ was demonstrated by Western Blot in 8 out of 8 cases of primary pre-B ALL. After incubation of primary pre-B ALL cells with different concentrations of CAL-101 (0.1-10 µM), dose-dependent downregulation of pAkt was observed. ALL cells were plated on immobilized VCAM1 (the counter-receptor for α4) or OP9 cells and treated with vehicle control (DMSO) or CAL-101 (10 µM) for 2 days. CAL-101 de-adhered ALL cells 69.5%±0.7% from VCAM1, and only 13%±0.5% from OP-9 cells, indicating an inhibition of the of the VCAM-1 and α4 interaction. ALL cells treated with CAL-101 (10 µM) for 5 days showed a marked decrease in viability as demonstrated by AnnexinV negative and 7AAD negative staining compared to DMSO-treated controls (66.2%±1.5% vs 87.9%±1.0%, p=0.0003) indicating that mono-treatment with CAL-101-decreases viability of ALL cells. Further studies addressing whether PI3Kδ inhibition is beneficial in the setting of combination chemotherapy for ALL in vitro and in vivo are ongoing.CONCLUSION: Taken together, our data demonstrate that the AKT pathway is affected by modulation of α4. PI3Kδ inhibition with CAL-101 de-adheres pre-B ALL cells from the α4-counter-receptor VCAM1, downregulates pAkt and induces apoptosis in ALL. Data derived from further studies will determine the potential of the FDA-approved PI3Kδinhibitor CAL-101 as a novel therapy for pre-B ALL. DisclosuresWayne:MedImmune: Honoraria, Research Funding, Travel Support Other; NIH: Co-inventor on investigational products with patents, Co-inventor on investigational products with patents Other.

Mechanistic rationale for targeting the unfolded protein response in pre-B acute lymphoblastic leukemia.

The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the endoplasmic reticulum (ER), regulates the expression and activity of molecules including BiP (HSPA5), IRE1 (ERN1), Blimp-1 (PRDM1), and X-box binding protein 1 (XBP1). These molecules are required for terminal differentiation of B cells into plasma cells and expressed at high levels in plasma cell-derived multiple myeloma. Although these molecules have no known role at early stages of B-cell development, here we show that their expression transiently peaks at the pre-B-cell receptor checkpoint. Inducible, Cre-mediated deletion of Hspa5, Prdm1, and Xbp1 consistently induces cellular stress and cell death in normal pre-B cells and in pre-B-cell acute lymphoblastic leukemia (ALL) driven by BCR-ABL1- and NRAS(G12D) oncogenes. Mechanistically, expression and activity of the UPR downstream effector XBP1 is regulated positively by STAT5 and negatively by the B-cell-specific transcriptional repressors BACH2 and BCL6. In two clinical trials for children and adults with ALL, high XBP1 mRNA levels at the time of diagnosis predicted poor outcome. A small molecule inhibitor of ERN1-mediated XBP1 activation induced selective cell death of patient-derived pre-B ALL cells in vitro and significantly prolonged survival of transplant recipient mice in vivo. Collectively, these studies reveal that pre-B ALL cells are uniquely vulnerable to ER stress and identify the UPR pathway and its downstream effector XBP1 as novel therapeutic targets to overcome drug resistance in pre-B ALL.

Galectin-3 in pre-B acute lymphoblastic leukemia

Galectin-3 in pre-B acute lymphoblastic leukemia

TRAIL Is Active Against Leukemia Stem Cells and Leukemia-Initiating Cells of Children with Acute Lymphoblastic Leukemia

Abstract 2953Cancer stem cells represent the most important target cells for anti-tumor therapy as they are uniquely capable to maintain tumor growth and to induce relapse. For sustained anti-tumor activity, anti-cancer therapy has to address and eliminate cancer stem cells. Unfortunately in acute lymphoblastic leukemia (ALL), cancer stem cells / leukemia stem cells (LSCs) can not be reliably defined by cell surface markers complicating studies on cancer stem cells. To overcome this obstacle, we used the functional definition of CSCs as surrogate and studied leukemia-initiating cells (LICs) by their name-defining capability to initiate leukemia in immune-compromised mice.TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family which induces apoptosis in a wide variety of tumor cells while sparing normal cells. TRAIL represents an important candidate anti-cancer agent which is currently tested in phase I and II clinical trials, yet its ability to target cancer stem cells is currently unknown.Here we tested the effect of TRAIL on LSCs and LICs using tumor cells from children with precursor B-cell acute lymphoblastic leukemia (pre-B ALL). Primary ALL cells were engrafted and passaged in NSG mice. Cells were freshly isolated from NSG mice and stimulated with TRAIL in vitro for 2 days and afterwards re-engrafted into mice in dilution curves upon limiting dilution transplantation assay comparing non-treated with TRAIL-treated cells and using about 25 mice per stimulation. After 12–16 weeks, mice were evaluated for leukemic engraftment by staining for leukemia cells in organs like bone marrow, spleen, liver and blood using FACscan analysis and immune histochemistry.In all 3 pre-B ALL samples tested, in vitro treatment with TRAIL prior to transplantation of cells into mice significantly reduced their engraftment capability. TRAIL disabled leukemic engraftment by > 95 % in all 3 samples. Accordingly, in vitro TRAIL treatment significantly increased the time to engraftment and completely disabled engraftment below a defined threshold.In a second, additive approach, TRAIL was used in a preclinical mouse model. Pre-B ALL samples were engrafted in NSG mice and leukemia-bearing mice were treated with TRAIL systemically at 7.5 mg / kg daily i.p. for 10 days. In this preclinical in vivo model, TRAIL treatment completely cured a proportion of animals harbouring patient-derived pre-B ALL xenografts.Taken together, TRAIL significantly disabled the leukemia-initiating function of LICs from patient-derived pre-B ALL xenografts in vitro and that TRAIL eliminated leukemia together with its LSCs in vivo. Although the methods used do not allow the study of signalling mechanisms, TRAIL might most probably have induced apoptosis in LICs and LSCs. Our data show that it is feasible, although technically demanding, to test the apoptosis sensitivity of LICs and LSCs.We conclude from these data that TRAIL constitutes an attractive future drug for treatment of ALL. Disclosures:No relevant conflicts of interest to declare.

Abstract B218: CBP/catenin antagonism inhibits self-renewal of acute lymphoblastic leukemia.

Abstract Background: Despite advances in chemotherapeutic treatment of acute lymphoblastic leukemia (ALL), drug resistance in ALL remains a major problem warranting new treatment strategies. Recent studies have demonstrated that survivin, a member of the inhibitor of apoptosis (IAP) family proteins, is upregulated in ALL of relapsed patients but not in drug-sensitive ALL. The expression of survivin depends on the formation of a complex between catenin (beta or gamma) and its co-activator CBP. T-cell factor (TCF)/catenin/CBP mediated transcription is involved in self-renewal of progenitor cells. However, a switch to TCF/catenin/p300 mediated transcription, which can be induced pharmacologically with ICG-001, a novel specific inhibitor of binding to the N-terminus of CBP, is described as critical to inhibit CBP-mediated self-renewal. We hypothesized that selective suppression of CBP/catenin signaling using ICG-001 offers a promising therapeutic principle to eradicate drug resistant ALL and showed previously that ICG-001 can sensitize patient-derived (primary) pre-B ALL cells to chemotherapy in vitro and in vivo. However, the underlying mechanism has not been described. Results: For this purpose, we initially determined expression of nuclear and cytosolic β- and γ-catenin expression in 13 primary ALL cases encompassing various cytogenetic aberrations by Western blot. Primary pre-B ALL cells were treated with and without ICG-001 and co-cultured with a murine stromal layer (OP9 cells) for 48 hours. Co-Immunoprecipitation showed that the binding of β- and γ-catenin with CBP, but not with p300, was inhibited by ICG-001 indicating that ICG-001 binds CBP and selectively disrupts β- and γ-catenin/CBP interaction. To determine the effect of ICG-001 on self-renewal in ALL, we performed a colony forming (CFU) assay with two primary ALL cells (LAX7R, SFO2) treated with single agent ICG-001 (10 m and 25 m). LAX7R showed a significant reduction with ICG-001 treatment (10 m and 25 m) compared with media only group (331.3× 42.8 and 57×13.5 vs 698.3× 68.4; p<0.05) in primary platings, and there were no colonies found in secondary platings of cells treated with ICG-001 (25 m), indicating that ICG-001 abrogates self-renewal of ALL cells. Similarly SFO2 cells showed no secondary colony counts with ICG-001 treatment (25 m) whereas control cells were replatable. These experiments were repeated three times in triplicate. Preclinical in vitro evaluation of five primary ALL cells treated continuously with ICG-001 (10μM- or, 25μM) or DMSO as vehicle control with and without chemotherapy consisting of Nilotinib for Philadelphia chromosome (Ph) positive ALL and Vincristine, Dexamethasone and L-Asparaginase for Ph negative ALL, that chemotherapy alone cannot eradicate primary ALL cells. However, in combination with ICG-001, chemotherapy led to the eradication of all five primary ALL cases, indicating that CBP/catenin antagonism can overcome drug resistance in ALL. Conclusion: Taken together, our data shows that targeting CBP/β- and γ-catenin by the small molecule inhibitor ICG-001 can abrogate primary ALL by inhibition of self-renewal of ALL cells. These findings provide the basis for a new treatment approach against drug resistant ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B218.