Survival Of pre-B Cells Research Articles

High Salt-Induced Hyperosmolality Reduces in Vitro Survival and Proliferation of Pre-B Cells

Aim: B cells of the adaptive immunity are critical for protection against the vast majority of pathogens through the production of specific antibodies. A number of signaling pathways and transcription factors control B cell development. Environmental factors, including diet, are also important in determining how B cell develop and function. Here, the effects of hyperosmolality induced by elevated salt on the survival, IL-7-induced proliferation and differentiation of pre-B cells were tested in vitro. 
 Material and Methods: The wk3 pre-B cell line generated from SLP65–/– mice was used. Hyperosmolality in the cell culture medium was created by increasing the salt concentration with the addition of 40 mM NaCl. Wk3 pre-B cells were cultured in standard (normal NaCl) and high salt (+40 mM NaCl) medium, followed by flow cytometric analysis.
 Results: It was found that hyperosmolality caused by high salt reduced survival and induced apoptosis in wk3 pre-B cells. In addition, hyperosmolality inhibited IL-7-induced proliferation of pre-B cells. Conversely, pre-B cells treated with high salt were able to differentiate normally into IgM+ immature B cells when IL-7 was removed. 
 Conclusion: These findings suggest that the hyperosmolar microenvironment induced by high salt may play a key role in B cell development in the bone marrow.

Interplay between PI3K/mTOR Signaling and IRE1a-XBP1 Promotes Survival of Pre-B NRASG12D all Cells Providing a Therapeutic Vulnerability for the “Undruggable” Driver RAS

Background Activating RAS mutations drive around 30% of pre-B cell acute lymphoblastic leukemias (pre-B ALL) and are particularly common in relapsed ALL with a consecutively poor outcome. Recently published data demonstrated the critical role of the unfolded protein response (UPR) network, namely its IRE1α-XBP1 axis, for the survival of pre-B ALL cells: High expression of XBP1 confers a poor prognosis in pre B-ALL. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. In this study, we aimed at identifying the molecular mechanism underlying pro-survival IRE1α-XBP1 signaling in RAS mutated pre-B ALL. Methods For a TET-ON inducible NRASG12D model in conditional Xbp1 knockout mice, we used interleukin 7 (IL-7)-dependent murine Mx1-Cre;Xbp1fl/fl pre-B cells transduced with a TET-ON inducible NRASG12D. We performed in vitro cell cycle and apoptosis assays with propidium iodide (PI) and annexin-V/PI. Furthermore, Western Blot and RT-qPCR were applied to analyze target gene expression. In a second approach, we focused on the signaling events following the blockade of RAS downstream targets using the MEK inhibitor PD0325901 and the dual PI3K/mTOR inhibitor BEZ235. We then assessed the efficacy of small molecule inhibition of IRE1α by MKC-8866 on XBP1 inactivation in RAS-mutated pre-B ALL cells either as a single treatment and in combination with the above mentioned drugs. Results We found the expression of Xbp1 significantly increased at the mRNA level with induction of NRASG12D. To determine the significance of Xbp1 in NRASG12D-driven pre-B ALL, we genetically deleted the IRE1α target Xbp1 using Cre-mediated deletion of Xbp1fl/fl in our mouse model of pre-B ALL. Genetic loss of Xbp1 significantly induced apoptosis (2.0-fold, p<0.0001) and caused cell cycle arrest (induction of G0/1, 1.7-fold, p=0.0003) along with an increase in the expression of CDK inhibitors, p21CIP1 and p27KIP1 at the protein level. Genetic ablation of Xbp1 abrogated IL-7 receptor (IL-7R) signaling by reducing the phosphorylation levels of STAT5-Y694 and JAK1-Y1022/Y1023. In an additional approach, we revealed that IL-7-deprived pre-B ALL cells reduce the mRNA expression of Xbp1s, indicating that Xbp1 acts as a downstream linchpin of the IL-7 receptor signaling pathway. Both IL-7-deprivation and genetic loss of Xbp1 increased the phosphorylation levels of ERK1/2-T202/Y204, AKT-S473 and the protein levels of NRASG12D and MAPK negative regulator DUSP6. Pharmacological inhibition of XBP1 activation using MKC-8866 resulted in similar effects on the expression of RAS downstream targets. We therefore tested MKC-8866 in combination with MEK inhibition by PD0325901 as a potential therapeutic strategy against pre-B ALL, which proved non-efficient. As a second option with therapeutic implications, we focused on the PI3K pathway which acts downstream of both the IL-7R and RAS signaling pathways. Strikingly, we observed that genetic ablation of Xbp1 (viable cells after 72 h, BEZ: 71.9 ± 9.0 vs BEZ+ Mx1-Cre;Xbp1fl/f: 10.0 ± 4.9) or pharmacological inhibition of its production with MKC-8866 (viable cells after five days, BEZ: 58.0 ± 6.8 vs BEZ+ MKC-8866: 13.3 ± 7.4) sensitizes pre-B ALL to dual inhibition of PI3K/mTOR with BEZ235. By applying the Bliss formula, we were able to show that BEZ235 in combination with MKC-8866 synergistically reduces the viability of RAS-mutated pre-B ALL cells. Gene expression analysis indicated that BEZ235 in combination with MKC-8866 fully blocked IL-7R signaling and caused an aberrant activation of Ras-Erk signaling. Targeting PI3K/mTOR signaling along with XBP1 inactivation increased expression of NRASG12D and its target DUSP6. In addition, we showed that combined therapy increased expression levels of p19Arf in RAS-mutated pre-B ALL, implicating cell senescence mediated by activated RAS signaling. Conclusion Our work strongly supports the hypothesis that XBP1 induces its positive effects on progression of pre-B ALL cells through the IL-7R signaling pathway. IL-7R signaling through its downstream effector XBP1 counteracts the RAS signaling pathway to promote leukemogenesis in pre-B ALL cells. Active XBP1 prevents the cytotoxic effects of BEZ235 in pre-B ALL cells, and hence targeting XBP1 in combination with dual PI3K/mTOR inhibition by BEZ235 appears as a promising targeted strategy against the "undruggable" driver RAS in NRASG12D-mutated pre-B ALL. Disclosures Brümmendorf: Janssen: Consultancy; Merck: Consultancy; Novartis: Consultancy, Other: travel, accommodation, expenses, Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Honoraria, Other: Travel, Accommodation, Expenses, Research Funding.

Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down syndrome

Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic "Philadelphia-like" ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remission and -relapse DS-ALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in the JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are "relapse driving." We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2pos DS-ALLs by loss-of-function aberrations in USP9X, a deubiquitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2pos, Philadelphia-like ALLs. Pharmacological or genetic inhibition of USP9X, as well as treatment with low-dose ruxolitinib, enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT "hypersignaling" may be beneficial to leukemic B-cell precursors. This finding and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.

PTEN as a tumor promoter

Because it enhances the survival of pre-B cells, PTEN could be targeted to treat pre-B acute lymphoblastic leukemia.

PAX5 promotes pre-B cell proliferation by regulating the expression of pre-B cell receptor and its downstream signaling.

PAX5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. Although previous studies have indicated that the Pax5-conditional-knockout mouse exhibited dedifferentiation of mature B cell and the development of aggressive lymphomas, the changes of Pax5 gene expressions in pre-B cells have not been analyzed. To understand the functional importance of Pax5 gene in the proliferation and survival of pre-B cells, we established a Pax5-knockdown model using 70Z/3 pre-B cell line. Pax5 knockdown 70Z/3 cells (70Z/3-KD cells) showed down-regulations of pre-BCR compounds such as CD19, BLNK, Id2 and λ5. The signaling via pre-BCRs was significantly diminished in the 70Z/3-KD cells, and this alteration was normalized by restored Pax5 gene expression. Loss of PAX5 reduced the growth rates in the 70Z/3-KD cells, compared to the mock cells. Meanwhile, the proliferation of pre-B cells was reduced by the knockdown of Pax5 gene. Moreover, further examinations showed that PAX5 was also activated in B cell acute lymphoblastic leukemia (B-ALL) as a cell proliferation enhancer. These findings suggested that pax5 is critically important for the proliferation and survival of pre-B cells.

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

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

Focal Adhesion Kinase Inhibitors Reverse the Stromal Adhesion Phenotype of Ikaros-Mutant B-ALL, Induce Apopotosis, and Synergize with ABL1 Tyrosine Kinase Inhibitors: A New Paradigm for Pathogenesis and Therapy of High-Risk B-ALL

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of precursor B-lymphocytes affecting both children and adults. Deletions and dominant-negative mutations in IKZF1, the gene encoding the Ikaros transcription factor, are found in ~85% of Ph+ B-ALL and in some cases of Ph– B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph– or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and imatinib have been added to chemotherapy regimens for Ph+ B-ALL, over half of these patients will still relapse, which correlates with residual disease burden in the bone marrow (BM) following induction therapy. Hence, new therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph– B-ALL.Using mice with a conditional Ikzf1 mutation (Ike5fl) where the recombined allele is similar to the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated recently that Ikaros DNA-binding function is required in the B-lymphoid lineage for transition from the large to small pre-B cell stage of differentiation, and that arrest at this stage of development can give rise to B-ALL (Joshi et al., Nat. Immunol. 2014;15:294). The survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK, downstream of integrins and growth factor receptors, which plays important roles in cancer stem cell biology, the tumor microenvironment and tumorigenesis. VS-4718 and VS-6063 (defactinib) are potent, orally bioavailable FAK inhibitors that inhibit tumor growth and metastasis in preclinical models, and are currently under evaluation in clinical trials in patients with various solid tumors. VS-6063 has demonstrated tolerability and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in phase I trials (ASCO, 2014). Here, we show that BCR-ABL1 cooperates with Ikzf1 mutation to accelerate B-leukemogenesis in mice. BCR-ABL1+ Ikaros-mutant B-ALLs exhibit stroma-mediated resistance to ABL1 TKIs, while the FAK inhibitors VS-4718 and VS-6063 are effective in blocking stromal adhesion and inducing apoptosis in both mouse and human Ikaros-mutant B-ALL samples.To test whether dysregulation of TK signaling cooperates with Ikzf1 mutation in the pathogenesis of high-risk B-ALL, we isolated BM B-lymphoid progenitor cells from wild-type (WT), IkE5fl/+CD2-Cre, and IkE5fl/flCD2-Cre donors, transduced them with BCR-ABL1 retrovirus and transplanted the cells into recipient mice. We observed a dramatic acceleration of precursor B-lymphoid leukemia induced by BCR-ABL1 in IkE5Δ/+ and particularly in IkE5Δ/Δ donor cells that correlated with a striking (~30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Relative to Ikzf1 WT BCR-ABL1+ leukemic cells, Ikzf1-mutant BCR-ABL1+ blasts showed significant resistance to imatinib and dasatinib that was dependent on the presence of OP9 stroma.The effect of FAK inhibition, using the FAK inhibitors VS-4718, VS-6062, and VS-6063 (Verastem), was first tested on murine B-ALL cells (genotypes Ikzf1 mutant, Ikzf1 mutant BCR-ABL1+, and Ikzf1 WT BCR-ABL1+) grown on OP9 stroma. FAK inhibitor treatment abolished stromal adhesion of Ikzf1-mutant B-ALL and induced apoptosis in non-adherent cells, but had little effect on Ikzf1 WT B-ALL cells. VS-4718 and VS-6063 were each synergistic with dasatinib in reducing the viability of Ikzf1-mutant BCR-ABL1+ B-ALL cells cultured on OP9 stroma. For primary human B-ALL samples grown on OP9 stroma, IKZF1-mutant cells were also more sensitive to FAK inhibitor treatment than WT IKZF1 WT B-ALL, with or without BCR-ABL1 expression.Collectively, these observations suggest a new model to explain the pathogenesis of high-risk B-ALL and its resistance to therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM LSCs. Inhibition of FAK signaling in Ph+ or Ph­–IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. Therefore, FAK inhibitors warrant further investigation for the treatment of high-risk IKZF1-mutant B-ALL patients. DisclosuresJoshi:Verastem: Research Funding. Yoshida:Verastem, Inc.: Research Funding. Paraskevopoulou:Verastem, Inc.: Research Funding. Zhang:Verastem, Inc.: Research Funding. Krause:Glycomimetics. Inc.: Research Funding. Shapiro:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Pachter:Verastem Inc.: Employment, Equity Ownership. Georgopoulos:Verastem, Inc.: Research Funding.

The survival and differentiation of pro-B and pre-B cells in the bone marrow is dependent on IL-7Rα Tyr449.

IL-7 is critical for murine T and B cell development and survival and plays a significant role in lymphoblastic leukemia in both humans and mice. We evaluated the role of the IL-7Rα Tyr(449) cytoplasmic SH2-binding motif in IL-7-mediated B cell development using a knock-in mouse with a Tyr to Phe mutation (IL-7Rα(449F/449F) mouse). IL-7Rα(449F/449F) and IL-7Rα(-/-) mice showed no defect in the number of pre-pro-B cells, although IL-7Rα(449F/449F) mice had decreased Ebf1 in pre-pro-B cells and impairment in B cell-committed CLPs. We identified that IL-7Rα Tyr(449) was critical for both pro-B and pre-B stages of development in the bone marrow. IL-7Rα(449F/449F) and IL-7Rα(-/-) mice had comparable precursor B cell defects, indicating that signaling from the IL-7Rα required this motif. Although the defect in IL-7Rα(449F/449F) pro-B cells was associated with loss of STAT5 activation and diminished expression of Mcl1, this was not rescued by overexpression of Bcl-2. IL-7Rα(449F/449F) and IL-7Rα(-/-) pre-B cells also showed defective cyto-Igμ and CD25 expression, associated with reduced levels of Rag1, Rag2, and Irf4. Pre-B cells from IL-7Rα(449F/449F) mice also failed to proliferate, perhaps as a result of the failure to rearrange Igμ. Our data suggest that IL-7Rα Tyr(449) was essential for IL-7Rα signaling in bone marrow B cell development and survival.

YB-1 Is Activated by IL-7 and Is Overexpressed in Pediatric Pre-B Acute Lymphoblastic Leukemia

Abstract 1468 Background:Increased YB-1 expression correlates with poor prognosis, drug resistance and metastasis in several different cancers including B cell lymphoma. Phosphorylation and nuclear localization of YB-1 in response to growth factors leads to increased survival through expression of proteins such as survivin and multidrug resistance protein 1. Until now, its role in leukemia has not been investigated. We hypothesized that YB-1 expression is aberrantly regulated in pediatric pre-B acute lymphoblastic leukemia (pre-B ALL), and that YB-1 may be activated downstream of IL-7. This cytokine facilitates the differentiation and survival of pre-B cells and has been implicated in the drug resistance of pre-B ALL. Methods:YB-1 and IL-7Ra protein expression was investigated by flow cytometry in normal pre-B cells (CD19+CD10+CD20−), and mature B cells (CD19+CD10−CD20+) as well as diagnostic and relapsed pre-B ALL (CD19+CD10+/−). Cell surface and cytoplasmic expression was quantified by mean fluorescent intensity (MFI). Bone marrow from healthy donors was used as a source of normal pre-B cells, while mature B cells were derived from PBMCs; leukemic cells at presentation and relapse were obtained following local IRB approval and informed consent. Activation of YB-1 downstream of IL-7 stimulation (25 ng/ml) was examined in pre-B ALL cell lines or NSG (NOD scid gamma) mice-expanded pre-B ALL by Western blotting using anti-phosphoYB-1(S012). Pre-B ALL cell lines used in these experiments were 697, 380, RCH and RS-4;11. Signaling pathways were investigated by pre-treatment of cells with pharmacological inhibitors followed by Western analyses. For the transient overexpression of YB-1, pEGFP or a pEGFP/YB-1 fusion protein was electroporated into freshly isolated mature B cells (which have a low basal expression of YB-1) and YB-1 and IL-7Ra expression was assessed by flow cytometry after 24 h. Results:While intracellular YB-1 expression was significantly higher in leukemia samples at presentation compared to normal pre-B cells, the highest YB-1 levels were found in relapsed pre-B ALL (see figure, part A). All examined pre-B ALL cell lines had levels comparable to those of the relapse samples. Similarly, surface IL-7Ra (CD127) levels (MFI medians; upper-lower range) were increased in onset (221; 150–286), and relapsed (1840; 651–2030) ALL compared to normal pre-B cells (528; 333–2673). (normal pre-B vs. leukemia at presentation, p<0.001, Mann-Whitney). Overexpression of YB-1-GFP in normal mature B cells resulted in increased expression of IL-7Ra (see figure, part B), suggesting an link between the YB-1 and IL-7 signaling pathways.Activated YB-1 is phosphorylated on S102 and relocated to the nucleus. Addition of IL-7 to pre-B ALL cell lines led to phosphorylation of YB-1 within 30 min. Similar results were shown for patient-derived, NSG mice-expanded pre-B ALL samples. Intracellular immunostaining using Imagestream technology (Amnis) showed that IL-7 treatment of pre-B ALL cell lines increased nuclear YB-1 levels 4-fold.As PI3K and MEK1 are involved in signaling downstream of IL-7, we investigated their role in YB-1 signaling in both pre-B ALL cell lines and NSG-mouse expanded pre-B ALL using pharmacological inhibitors. Western analyses showed that inhibition of PI3K using LY294002 did not prevent IL-7-mediated phosphorylation of YB-1 but the MEK1 inhibitor U0126 did, indicating the involvement of MAPK (see figure, part C). Conclusion:We show that YB-1, which is highly expressed in pediatric pre-B ALL compared to normal pre-B cells, is expressed at even higher levels after relapse. We demonstrate a link between the YB-1 and IL-7 signaling pathways which could offer a novel target for the treatment of refractory leukemia. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

C-Myb interacts with the glucocorticoid receptor and regulates its level in pre-B-acute lymphoblastic leukemia cells

Glucocorticoid (GC) hormones are used in the treatment of hematopoietic malignancies. When the GC binds to the glucocorticoid receptor (GR) protein, c-Myb and GR are recruited at the Glucocorticoid Response Unit in the DNA. Here we demonstrate that c-Myb interacts with the GR and that decreasing c-Myb amounts reduces the levels of GR transcripts and protein in 697 pre-B-acute lymphoblastic leukemia (ALL) cells. Furthermore, the auto-upregulation of GR promoter 1C and promoter 1D is blunted at reduced c-Myb levels. Taken together, these data show that c-Myb is a direct, key regulator of the GR. Unexpectedly, the reduction in c-Myb levels increased the sensitivity of the cells to steroid-mediated apoptosis. This was because the reduction in c-Myb itself decreases cell viability, and the residual GR remained above the threshold needed to trigger apoptosis. These studies show the mutual importance of c-Myb and the GR in controlling survival of pre-B ALL cells.

Central Nervous System Involvement in a Xenograft Model of Pre-B Acute Lymphoblastic Leukaemia Is Associated with Dysfunctional CXCR4 Expression and Upregulation of the Neurotropic Chemokine Receptors CCR6 and CX3CR1,

Abstract 3449Despite huge advances in the treatment of paediatric acute lymphoblastic leukaemia (ALL) challenges remain. Disease in the central nervous system (CNS) continues to pose difficulties in diagnosis, prevention and treatment. Understanding the biological mechanisms of leukaemic cell entry into the CNS should allow better detection and monitoring of leukemia and may identify novel therapeutic targets for resistant and relapsed disease. We hypothesize that leukaemic cell dissemination to the CNS is associated with the abnormal expression of molecules governing physiological leukocyte trafficking i.e. chemokine receptors, selectins and integrins. To address this hypothesis we have developed a xenograft model of CNS disease using IV tail vein injection of human leukaemic cell lines and primary cells into immunodeficient (NOD/SCID/IL2R gamma null) mice.Pre-B leukaemic cell lines were seen to differ in their capacity to home to the CNS. Using Taqman low density array plates quantitative expression of a panel of chemokine receptors, selectins and integrins was compared between these cell lines. Rapid onset of CNS disease was associated with significantly higher expression of P-selectin glycoprotein ligand 1 and the chemokine receptor CCR6 both known to be essential for blood:CSF barrier transit of leukocytes (Kivisakk et al PNAS 2003, 100, 8389–8394, Reboldi et al Nat Immunology 2009, 10, 514–523). Other genes upregulated in CNS homing cells included (1) the integrins alphaM beta2 and beta 7 (2) ICAM-1 and −3 and (3) the chemokine receptors CCR1, CCR7 and CXCR3.Interestingly the chemokine receptor CXCR4 showed down-regulation when measured by qPCR and flow cytometry in CNS homing cells, with levels of receptor expression inversely proportional to the rapidity of onset of CNS disease. Furthermore in vitro studies showed that the migratory response of CXCR4 to its ligand CXCL12 was blunted or absent in cell lines which produced a more rapid onset of CNS disease. Two of the cell lines were Philadelphia positive, raising the possibility that p190 bcr-abl could be interfering with CXCR4 signalling or receptor levels as previously demonstrated for the p210 bcr-abl fusion protein (Geay et al Cancer Res 2005, 65, 2676–2683). Although non-migratory cells had higher levels of bcr-abl expression than migratory cells the blunted responses could not be reversed by treatment with the bcr-abl inhibitor imatinib. CXCR4 mutations were excluded by direct sequencing. Since functional CXCR4-CXCL12 interactions are known to be important for retention of cells in the bone marrow microenvironment (Ma et al, Immunity 1999, 10, 463–71), disruption of this interaction may be a necessary pre-requisite for cell migration to other sites.To examine potential micro-environmental influences on gene expression patterns in vivo, cells were retrieved from the CNS, bone marrow, liver, spleen and kidneys of engrafted mice using anti-human CD19 magnetic bead sorting and species specific primers for qPCR. Cells derived from the CNS had higher levels of CCR6 and the neurochemokine CX3CR1 (fractalkine receptor) compared to the original cell line in vitro and cells retrieved from other sites. Increased CCR6 may represent sub-clonal selection of CCR6 high expressors during transit across the BCSFB. Fractalkine and its receptor are highly expressed in the central nervous system and are important for maintenance of microglial-neuronal communication with fractalkine activating pro-survival signaling pathways in cells bearing its receptor (Meucci et al PNAS 2000, 97, 8075–8080). This provides a possible mechanism by which fractalkine expression would provide a competitive advantage to cells and allow survival of pre-B cells in this normally hostile microenvironment.In conclusion, we present a xenograft model of CNS leukemia and its utilization to identify increased CCR6 and P-selectin glycoprotein ligand 1 expression and reduced CXCR4 expression (and/or function) as candidate mechanisms by which leukaemic pre-B cells cross the blood:CSF barrier. In addition we propose that the Fractalkine receptor CX3CR1 may act as a potential pro-survival mechanism for pre-B cells residing in the CNS. As well as shedding light on the biology of CNS disease in pre-B cell ALL these molecules may be valid novel therapeutic targets for resistant or relapsed CNS disease. Disclosures:No relevant conflicts of interest to declare.

CRLF2 and JAK2 Mutations: Occurrence and Function In Pre-B ALL Cell Lines

AbstractAbstract 5109Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells promotes early B-cell development and activates dendritic cells. It has recently been reported that a subset of B-cell precursor acute lymphoblastic leukemia (pre-B ALL) overexpresses the TSLP receptor CRLF2. CRLF2 overexpression is linked to translocations between sex chromosomes – localizing CRLF2 – and the immunoglobulin heavy chain locus on chromosome 14, or to an interstitial deletion on the gonosomes. Both events, translocation and deletion juxtapose CRLF2 to a different promoter (IgH or P2RY8). Performing quantitative real-time PCR we tested pre-B ALL and acute myeloid leukemia (AML) cell lines for overexpression of CRLF2. AML cell lines were included in the screening because we knew from an earlier TSLP project that the AML cell line MUTZ-3 is TSLP-responsive, and thus positive for the cytokine receptor. Three of 63 (5%) pre-B ALL cell lines tested (INC, MHH-CALL4, MUTZ-5) overexpressed CRLF2 mRNA. CRLF2-high cell lines carry a t(14;Y). With respect to the 58 AML cell lines tested: some expressed CRLF2 mRNA, but none of them rivalled the aforementioned pre-B cell lines. Pre-B ALL cell lines show the association between chromosomal CRLF2 aberrations and JAK2 pseudokinase domain mutations that has been described for primary pre-B ALL cells: cell lines MHH-CALL4 (JAK2I682F) and MUTZ-5 (JAK2R683G) and – newly described - also the CRLF2-high pre-B ALL cell line INC express a mutated version of Janus kinase 2 (JAK2R683G). We established a PCR based assay system that allowed for the rapid detection of the JAK2R683G mutation: none of the CRLF2-low or –negative pre-B ALL cell lines exhibited this mutation. All three CRLF2-high/JAK2mu cell lines showed high phosphorylation levels of the JAK2 downstream target STAT5. Inhibition of the JAK kinase led to dephosphorylation of STAT5. However, repression of 3H-thymidine uptake and induction of apoptosis by inhibition of the JAK2/STAT5 pathway was weaker in the JAK2mu pre-B ALL cell lines than in the JAK2V617F positive essential thrombocythemia-derived cell line SET-2. Provided that these results reflect the situation in primary cells, mutated JAK2 seems to be of lesser importance for growth and survival of pre-B ALL cells than for cells from myeloproliferative neoplasms. The CRLF2-high/JAK2mu cell lines INC, MHH-CALL4 and MUTZ-5 are promising model systems for the study of the roles of high-level CRLF2 expression and of JAK2 mutations in pre-B ALL. Disclosures:No relevant conflicts of interest to declare.

All oligosaccharide moieties of the μ chains in the pre-BCR are of the high-mannose type

Although it is well established that pre-BCR signaling governs proliferation and differentiation during B cell development, the components of the pre-BCR that are important for signaling are a matter of controversy. It has been suggested that signaling by the μ heavy chains of the pre-BCR induces survival and differentiation of pre-B cells, while the λ5 part of the pre-BCR is essential for proliferation and clonal expansion. However, the mechanism by which pre-BCR μ chains initiate differentiation signals is not clear. Using two variants of a murine B-lymphocyte cell line that differ only in surface expression of either BCR or pre-BCR, we demonstrated that surface μ chains in the pre-BCR are of the high-mannose type only, while those in the BCR are of the complex type. It is hypothesized that mannose-specific lectin-like molecules on accessory cells or in solution may function as the non-antigen ligand that triggers the pre-BCR.

Ptch1 Regulates the Bone Marrow Cell Niche Critical for Survival of Pre-B Cells but Not Hematopoietic Stem Cells

Activation of the Hedgehog (Hh) signalling pathway by loss of function mutations of the Ptch1 receptor promotes stem or progenitor cell proliferation in several cell types, most notably the basal cells of the skin and granule cells of the cerebellum. We have intercrossed MxCre transgenic mice with conditional Ptch1 knockout mice to study the effects of deleting Ptch1 on adult hematopoiesis, with the hypothesis that loss of Ptch1 would activate the Hh pathway leading to increased hematopoietic stem cells (HSC). Within 4 weeks after deletion of Ptch1 with administration of poly(I:C), MxCrePtch1-null mice developed apoptosis of bone marrow pre-B cells and double positive thymocytes. Overall, MxCrePtch1-null mice have 10-fold less pre-B cells and thymocytes. MxCrePtch1-null mice also develop a 3-fold increase in lineage negative c-kit+ Sca-1+ (LKS) bone marrow cells, a cell fraction enriched for HSCs. Despite increased numbers of LKS, loss of Ptch1 did not increase the numbers of HSCs as measured by competitive repopulation assays. MxCrePtch1-null mice also developed typical Ptch1-related tumours including basal cell carcinomas and cerebellar tumours, which was consistent with the ability of the MxCre transgene to delete loxP-flanked genes in cell types other than hematopoietic cells. To determine if the hematopoietic changes observed in the MxCrePtch1-null mice were cell intrinsic or due to loss of Ptch1 on cells of the microenvironment, we intercrossed conditional Ptch1 mice with hematopoietic specific Cre transgenic mice. Surprisingly, HSC-specific deletion of Ptch1 using tamoxifen-inducible SCLert(2)Cre mice did not lead to any increase in LKS numbers. Similarly, lymphoid specific deletion of Ptch1 with the B-cell specific CD19Cretransgene or the T-cell specific LckCre transgene did not lead to any lymphoid defects. The lack phenotype in hematopoietic-specific Ptch1-null mice indicates that Ptch1 is redundant on hematopoietic cells including HSCs. Furthermore, the lack of phenotype also suggests that the defects observed in the MxCrePtch1-null mice were due to loss of Ptch1 in the microenvironment. To prove that Ptch1 regulates the hematopoietic microenvironment, we performed reciprocal transplant experiments whereby lethally irradiated MxCrePtch1- null mice were reconstituted with wild-type bone marrow cells. Remarkably, wild-type hematopoiesis grown within the MxCrePtch1-null microenvironment developed the identical hematopoietic defects with increased LKS and apoptosis of pre-B cells and thymocytes. Conversely, the MxCrePtch1-null hematopoietic defects could be completely rescued by transplant into lethally irradiated wild-type mice. Histological examination of bones from MxCrePtch1-null mice showed marked alterations in trabecular and cortical bone. Given the recent demonstration that the Hh pathway regulates adult bone homeostasis, we hypothesize that the increased LKS and loss of pre-B cells observed in Ptch1-null mice are secondary to changes within the bone marrow cell niche.

A Unique Role for the λ5 Nonimmunoglobulin Tail in Early B Lymphocyte Development

Precursor BCR (pre-BCR) signaling governs proliferation and differentiation of pre-B cells during B lymphocyte development. However, it is controversial as to which parts of the pre-BCR, which is composed of Igmu H chain, surrogate L chain (SLC), and Igalpha-Igbeta, are important for signal initiation. Here, we show in transgenic mice that the N-terminal non-Ig-like (unique) tail of the surrogate L chain component lambda5 is critical for enhancing pre-BCR-induced proliferation signals. Pre-BCRs with a mutated lambda5 unique tail are still transported to the cell surface, but they deliver only basal signals that trigger survival and differentiation of pre-B cells. Further, we demonstrate that the positively charged residues of the lambda5 unique tail, which are required for pre-BCR self-oligomerization, can also mediate binding to stroma cell-associated self-Ags, such as heparan sulfate. These findings establish the lambda5 unique tail as a pre-BCR-specific autoreactive signaling motif that could increase the size of the primary Ab repertoire by selectively expanding pre-B cells with functional Igmu H chains.

Thymic Stromal-Derived Lymphopoietin Induces Proliferation of Pre-B Leukemia and Antagonizes mTOR Inhibitors, Suggesting a Role for Interleukin-7Rα Signaling

Understanding the pathogenesis of leukemia in the context of lymphopoiesis may reveal novel therapeutic targets. Previously, we have shown that mTOR inhibitors (MTI) show activity in vitro and in preclinical models of both human and murine precursor B acute lymphoblastic leukemia (pre-B ALL), inhibiting cell proliferation and inducing apoptosis. These MTI-mediated effects can be reversed by interleukin-7 (IL-7), an important regulator of early B-cell development. This observation led us to examine the contribution of signaling via the IL-7Ralpha chain, which is shared by the receptor complexes of IL-7 and thymic stromal-derived lymphopoietin (TSLP). TSLP is closely related to IL-7 and active in lymphopoiesis, but an effect of TSLP on leukemia cells has not been described. We examined the effect of TSLP on pre-B ALL cells and their response to MTIs. Here, we show that TSLP stimulates proliferation of pre-B ALL cell lines. TSLP also partially reverses the effects of MTI on proliferation, apoptosis, and ribosomal protein S6 and 4E-BP1 phosphorylation in cell lines, with similar biological effects seen in some primary human lymphoblast samples. These data show that TSLP can promote survival of pre-B ALL cells and antagonize the effects of MTIs. These findings suggest that IL-7Ralpha chain is responsible for transducing the survival signal that overcomes MTI-mediated growth inhibition in pre-B ALL. Thus, further exploration of the IL-7Ralpha pathway may identify potential therapeutic targets in the treatment of ALL. Our data illustrate that growth-factor-mediated signaling may provide one mechanism of MTI resistance.

The Wnt Pathway Modulates Expression of Growth and Survival Genes in Acute Lymphoblastic Leukemia Cells.

Wnt proteins are important bone marrow-derived growth factors known to support normal hematopoietic progenitor and stem cell development. Here we report that B cell progenitor acute lymphoblastic leukemia (pre-B ALL) cells express Wnt proteins, including Wnt-2b in 33%, Wnt-5a in 42%, Wnt-10b in 58% and Wnt-16b in 25% of cases. The Wnt receptors, Frizzled (Fz)-7 and -8 were also expressed in most cases while Fz-3, -4 and -9 were occasionally detected. Stimulation of pre-B ALL cells with Wnt-3a activated canonical Wnt signaling with increased expression and nuclear translocation of β-catenin. This resulted in a 1.7 to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. Wnt-3a also significantly increased the survival of pre-B ALL cells under conditions of serum deprivation. To determine the mechanisms involved we examined the effects of Wnt-3a on gene expression using the leukemic pre-B ALL cell line NALM6 and a cancer specific microarray (GEArray® OHS-802), which contains 440 known cancer genes. Expression of 83 genes (19%) could be detected on the array. Exposure to Wnt-3a for 24 hours resulted in increased (>1.5 fold) expression of 29 genes and reduced (<50% of control) expression of 3 genes. The most highly regulated genes in response to Wnt-3a were MYBL2, E2F1, CD10, VDAC1, CDC25B (upregulated) and TRAIL-R2 (downregulated). Using qRT-PCR, we confirmed regulation of these genes in NALM6 cells and/or in another leukemic cell line LK63. These genes play important roles in the control of cell cycle (MYBL2, E2F1 and CDC25B), apoptosis (VDAC1 and TRAIL-R2) and motility (CD10) in cancer cells. Our results suggest that Wnt signalling regulates cell growth and proliferation in leukemic cells by modulating the expression of a number of genes. To our knowledge this is the first study examining the gene expression profile following Wnt stimulation in leukemic cells and potentially identifies new therapeutic targets for treatment.

Pre-B Cell Receptor Assesses the Quality of IgH Chains and Tunes the Pre-B Cell Repertoire by Delivering Differential Signals

It is well understood how a variety of Ig H and L chains, components of BCR, are generated in the DNA level during B cell development. However, it has remained largely unknown whether and how each component is monitored for its quality and selected before the assembly into the BCR. Here we show that muH chains produced by pre-B cells display a wide spectrum of ability to form the pre-BCR, which is composed of muH and surrogate light (SL) chains and is crucial for B cell development. The level of surface pre-BCR expression varies among pre-B cells, depending on the ability of their muH chains to pair with SL chains. The higher the level of pre-BCR expression by pre-B cells, the stronger their pre-BCR signaling, and the better they proliferate and differentiate. Thus, the extent of survival, proliferation, and differentiation of individual pre-B cells is primarily determined by the SL-pairing ability of their muH chains. Furthermore, IgH chains with higher potential to assemble with IgL chains appear to be positively selected and amplified through the assessment of their ability to pair with SL chains at the pre-BCR checkpoint before the assembly into the BCR. These results indicate that the pre-BCR assesses the quality of muH chains and tunes the pre-B cell repertoire by driving the preferential expansion and differentiation of cells with the higher quality of muH chains.

Disruption of the Shc/Grb2 Complex during Abelson Virus Transformation Affects Proliferation, but Not Apoptosis

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces pre-B-cell transformation. Signals emanating from the SH2 domain of the protein are required for transformation, and several proteins bind this region of v-Abl. One such protein is the adaptor molecule Shc, a protein that complexes with Grb2/Sos and facilitates Ras activation, an event associated with Ab-MLV transformation. To test the role this interaction plays in growth and survival of infected pre-B cells, dominant-negative (DN) Shc proteins were coexpressed with v-Abl and transformation was examined. Expression of DN Shc reduced Ab-MLV pre-B-cell transformation and decreased the ability of v-Abl to stimulate Ras activation and Erk phosphorylation in a Raf-dependent but Rac-independent fashion. Further analysis revealed that Shc is required for v-Abl-mediated Raf tyrosine 340 and 341 phosphorylation, an event associated with Erk phosphorylation. In contrast to effects on proliferation, survival of the cells and activation of Akt were not affected by expression of DN Shc. Together, these data reveal that v-Abl-Shc interactions are a critical part of the growth stimulatory signals delivered during transformation but that they do not affect antiapoptotic pathways. Furthermore, these data highlight a novel role for Shc in signaling from v-Abl to Raf.

The BCR-ABL1 Kinase Interferes with Pre-B Cell Receptor Signal Transduction in B Cell Precursor Leukemia Cells.

The BCR-ABL1 kinase expressed in B cell precursor leukemia drives malignant transformation of human pre-B cells. To identify novel target genes of BCR-ABL1-mediated transformation and to elucidate how the oncogenic BCR-ABL1 kinase may interfere with normal pre-B cell receptor signaling, we compared genome-wide gene expression of BCR-ABL1+ B cell precursor leukemia cells with their normal counterpart.While pre-B cell receptor signals are critical for differentiation and survival of normal pre-B cells, nearly all components of pre-B cell receptor-dependent signal transduction were downregulated in the leukemia cells. Surprisingly, BCR-ABL1+ leukemia cells mostly even do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH gene rearrangement nonfunctional. However, even BCR-ABL1+ leukemia cells harboring a functional VH gene rearrangement are unresponsive to pre-B cell receptor engagement. Independently from engagement, BCR-ABL1 kinase activity induces activation of PLCg1, resulting in an autonomous oscillatory Ca2+ signaling. BCR-ABL1 kinase activity is also linked to the expression of a truncated isoform of the pre-B cell receptor-associated linker molecule SLP65, which acts as a tumor suppressor in mice. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 show responsiveness to antigen receptor engagement and express only full-length SLP65. These cells differentiate into immature B cells expressing Ig light chains.We conclude that BCR-ABL1 interferes with pre-B cell receptor signal transduction in B cell precursor leukemia cells, while its inhibition reconstitutes the selection for functional (pre-)B cell receptor signaling. Hence, a functional pre-B cell receptor signal-transduction cascade may provide these cells with a potential mechanism to escape apoptotic cell death induced by inhibition of BCR-ABL1.Figure Legend:BCR-ABL1+ leukemia cells were incubated for 48 hours in the presence or absence of 10 μmol/l STI571. Differentiating subclones among the STI571-treated cells replace their pre-B cell receptor by a B cell receptor and were enriched for surface IgM-expression by MACS. [Display omitted]