ALBERT

Description: PAX5 is a transcription factor acting both as an activator and a repressor, known to be frequently altered (30% of the cases) in B Cell Precursor Leukemia (BCP-ALL). PAX5 aberrations include point mutations, deletions, amplifications and translocations with several partner genes. All the originated fusion genes retain the PAX5 DNA binding domain but acquire the regulatory regions of the partner gene, showing a dominant negative mechanism on endogenous PAX5. However, the function of PAX5 fusion proteins and their common mechanisms of leukemogenesis are poorly understood. We have established an in vitro model in which wild-type murine pre-BI cells were transduced with the most recurrent PAX5 fusion gene, PAX5/ETV6. Gene expression profiling of transduced PAX5/ETV6 pre-BI cells showed a significantly altered transcription profile compared to empty vector control. Interestingly, pathway analyses revealed that many of the genes implicated in the B-cell receptor (BCR) assembly and signaling were repressed by PAX5/ETV6. In addition, the downstream signaling of the BCR was non-functional through a block in IgM heavy chain rearrangement. Aim of the current study has been to elucidate the signaling pathway activated either in PAX5/ETV6 transduced pre-BI cells and in primary cells from patients carrying different PAX5 translocations, in order to overcome the absence of a functional BCR expression. Interestingly, among the top-ranked over-expressed genes in PAX5/ETV6 transduced pre-BI cells, the PAX5 repressed target gene LCK was up-regulated both at basal level (mean fold change by RQ-PCR in 3 different pre-BI lines: 1.79, p

Description: Oncogenic lesions in hematopoietic progenitor cells give rise to B-cell or myeloid malignancies. While often transformed by the same oncogenes, B-cell and myeloid leukemias markedly differ in biological and clinical characteristics. Our metabolic analyses revealed that B-cell-unlike myeloid-leukemia cells are massively restricted in their glycolytic capacity. Low glycolytic reserves in B cells resulted in a state of chronic energy depletion and engaged the energy sensor LKB1-AMPK. Myeloid cells strongly activated glucose transport through insulin receptor (INSR)-AKT signaling and lacked activity of LKB1-AMPK, reflecting energy abundance. Conversely, B-cells lacked INSR-AKT signaling and were critically dependent on LKB1-AMPK-mediated glucose uptake. Cre-mediated deletion of Lkb1 caused acute glycolytic exhaustion and cell death in B-lineage but increased glycolysis, energy levels and proliferation in myeloid leukemia. C/EBPa-mediated conversion of B-cell into myeloid identity reversed the detrimental effects of Lkb1-deletion and restored glycolysis, energy levels and survival of B→myeloid reprogrammed cells. In 〉80% of B-lineage leukemia cases, we found genetic lesions of transcription factors (e.g. deletion of PAX5, IKZF1, rearrangement of MLL) that caused a B→myeloid lineage shift. While previously of unknown functional significance, these lesions relieved B-cell-specific transcriptional repression of molecules that mediate glucose uptake and utilization (INSR, GLUT1, HK2, G6PD) and amplified glycolytic energy supply for transforming oncogenes. Likewise, glucocorticoid receptor (NR3C1)-mediated inhibition of glucose uptake and glycolysis was strictly dependent on a B-lymphoid transcriptional program. B→myeloid lineage conversion abolished NR3C1 expression and activity, which provides a mechanistic explanation for the empiric finding that glucocorticoids are highly active in the treatment of B-cell-but not myeloid malignancies. In conclusion, B-cell-specific restriction of glycolytic energy supply represents a previously unrecognized metabolic barrier against malignant transformation and reveals LKB1-AMPK as a novel target for the treatment of human B-lineage leukemia. Disclosures No relevant conflicts of interest to declare.

Description: 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. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2482 Introduction. PAX5 is a transcription factor with both activation and repression functions, essential for B-cell development. Recently, it has been found as frequent target of abnormalities in B-cell precursor ALL, showing point mutations, deletions or involvement in chromosomal translocations. The functional role of these lesions is still poorly understood. In previous experiments in mouse pre-BI cells, we showed that the PAX5/TEL protein acts as an aberrant transcription factor with repressor function, causing a block on B-cell differentiation, short-term IL-7 independence and resistance to the anti-proliferative and pro-apoptotic effects of TGFbeta1. Moreover, PAX5/TEL enhances cell migration towards CXCL12, with the over expression of CXCR4. Aim. The aim of the present study was to comprehensively understand how PAX5/TEL affects the transcription process and eventually interferes with PAX5 and TEL pathways and how these modulate cellular processes. Methods. We analyzed gene expression profiles in pre-BI cells transduced either by MIGR-PAX5/TEL-IRES-GFP or by MIGR-GFP (Affymetrix Gene Chip technology, Mouse array 430A 2.0). Validation of Differentially Expressed Genes has been performed by quantitative RQ-PCR and FACS analyses. In vitro adhesion assays have been performed on VCAM1-coated slides. Results. PAX5/TEL significantly modulated the transcription process: among 340 differentially expressed genes, 61% were down- and 39% up-regulated. Both up and down-regulated genes encompass numerous PAX5-target genes; in particular, PAX5/TEL represses genes which are normally activated by PAX5, and, vice versa, it activates genes physiologically repressed by PAX5. Moreover, gene function classification analyses suggested that PAX5/TEL modulates molecules which are related to fundamental cellular processes, such as phosphorylation, transcription, B cell receptor signaling, as well as adhesion. In particular, we demonstrated the modulation of surface antigens responsible of extra cellular binding as well as the modulation of intracellular molecules involved in the signaling of adhesion regulation, such as CD44, SDC4, EDG1, NEDD9, BCAR3, PLEKHA2, SPHK1. Moreover, in vitro adhesion assays on VCAM1-coated slides showed a significant reduction of adhesion capacities in PAX5/TEL positive cells. In agreement with our previous results, which showed down-regulation of CD19, BLNK/SLP-65 and MB-1/CD79a, both genes involved in BCR signaling, we demonstrated the additional repression of numerous key molecules fundamental for this pathway, such as SIGLECG/CD22, IRF4, LCP2/SLP-75, SLAMF6/LY108, PLEKHA2, PRKD2, IKZF2, IKZF3. Furthermore, we functionally validated the impairment of BCR-signaling and demonstrated that PAX/TEL transduced pre-BI cells are completely blocked in IgM protein expression, loosing the ability to complete the VDJ rearrangement and consequently express the m-chain on the cells surface, compared to the control cells. Conclusions. These analyses further sustain the role of PAX5/TEL as an aberrant transcription factor. Its effect on endogenous PAX5 does not represent a classical dominant negative role; indeed, we defined this effect as an ‘opposite dominance’, since PAX5/TEL caused the up-regulation of PAX5-repressed genes and, vice versa, the down-regulation of PAX5-activated targets. The biological consequences of this aberrant transcriptional activity are the impairment of B cell receptor signaling and the reduced adhesion capacity, both fundamental processes in B-cells, potentially involved in tumor transformation and in leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL) is characterized by the translocation t(9;22), resulting in the BCR-ABL1 fusion gene. It occurs in 2-3% of pediatric ALL and is associated with poor prognosis. Despite the introduction of tyrosine kinase inhibitors (TKIs), such as Imatinib, resulting in a significant increase in the cure rate, a consistent number of patients show resistance to treatment and subsequently relapse. In an adult cohort, more than 70% of relapses during TKI treatment involve selection of tyrosine kinase domain (TKD) mutations. In order to better understand the mechanism of Imatinib resistance in pediatric Ph+ ALL, we selected as our cohort a subgroup of patients treated with Imatinib on top of chemotherapy, according to the EsPhALL clinical trial and enrolled in Italy from January 2004 to December 2014 by the AIEOP study group. Patients (n=10) either didn't achieve remission or experienced early relapse by the end of 2014. Patients' samples have been screened for mutations in the ABL1 tyrosine kinase domain (TKD). Nine out of ten BM samples collected at relapse showed wild type TKD. Accordingly, no subsequent relapses showed the appearance of an ABL1 mutated clone. Only one patient carried the tyrosine 243 to phenylalanine substitution (Y253F) at the first and second relapses, but not at diagnosis, demonstrating the expansion of a resistant minor clone at diagnosis, following TKI treatment. In addition, we monitored the BCR-ABL1 transcript level by RQ-PCR at diagnosis, relapse and multiple follow up points. In nine out of ten patients the expression of BCR-ABL1 was highly positive at the time of the relapse, and in five out of six cases the increase of the transcript level was predictive of a subsequent clinical relapse. Moreover, we investigated the cooperative copy number alterations by MLPA analysis in 6 matched diagnosis and relapse(s) samples. MLPA data showed a heterogeneous scenario of clonal evolution. In three patients, the relapse clone shared only a minor genetic identity with the predominant clone at diagnosis and wasn't the product of its direct evolution. In one case, the clone at relapse was identical to that at diagnosis, while in two cases the predominant clone at relapse was the product of the direct clonal evolution of the main clone at diagnosis. In 4 cases, the dynamics of clonal evolution were confirmed by the analyses of the IG/TCR rearrangements. In conclusion, our data depict a very different scenario of resistance in childhood Ph+ ALL compared to the adult cohort. Indeed, Imatinib resistance doesn't seem to be dependent on ABL1 mutations in pediatric Ph+ ALL. Moreover, the MLPA results contribute to the description of clonal heterogeneity in Ph+ ALL, and may partially explain the different response to treatment. Disclosures No relevant conflicts of interest to declare.

Description: PAX5, located on 9p13, belongs to the PAX gene family and encodes for a transcription factor essential for B lymphoid cell commitment. It functions both as a transcriptional activator and repressor of different target genes involved in lineages development. PAX5 has been recently reported to be target of aberrancies (including point mutation, deletions, and gene fusions), in approximately 30% of pediatric patients affected by BCP-ALL, the most frequent leukemia subset in children. Translocations are estimated to occur at an incidence of 2-3%, with a variety of partner genes, encoding for transcription factors (TEL, PML, FOXP1), kinases (JAK2), structural proteins (ELN, POM121) or molecules of unknown function (C20orf112, AUTS2). We performed a FISH-based study on an Italian cohort of BCP-ALL patients having 9p13 chromosomal rearrangement (as a hallmark of PAX5 rearrangement), and we identified novel PAX5 partner genes. Two pediatric patients were harboring a t(7;9)(q11.2;p13.2) with a PAX5/AUTS2 fusion transcript, thus confirming its recurrent alteration in pediatric B-ALL. Three novel partner genes of PAX5 were identified by FISH. SOX5 was found as a PAX5 partner in a pediatric patient harboring a dic(9;12)(p13;p13) chromosome. A further patient, showing a t(9;12)(p13;q34) translocation, revealed PAX5 as fused to a novel transcript isoform of CHFR, a gene widely expressed in a library of normal tissues. A third partner was identified in an adult B-ALL case, which showed a deletion within the short arm of chromosome 9, leading to the fusion of PAX5 to MLLT3. A fourth PAX-rearranged case, involving POM121C (different from the already described POM121) as fused to PAX5 in a t(7;9)(q11;p13) translocation, was identified by a RNAseq approach on BCP-ALL cases without known prognostic features. The breakpoint on chromosome 7q11 is similar to the one associated with PAX5/AUTS2. An accurate FISH analysis was performed on bone marrow cells of all cases to dissect the genomic breakpoints and the structure of the rearrangements. The fusion genes were cloned by 5’ and/or 3’ RACE PCR, confirmed by sequencing and verified by RT-PCR with specific primers on the source material. PAX5-translocated cases were further characterized by genome-wide Single Nucleotide Polymorphism array. Interestingly, Copy Number Variation analysis showed that a limited number of cooperative genetic lesions were present in addition to the translocation event, thus suggesting a primary role of the PAX rearrangement in leukemogenesis. We therefore hypothesize that PAX5 alterations may represent single genetic aberration events in a simple background, rather than being part of a complex scenario of cooperating genetic lesions involved in leukemogenesis. A common pathway for all PAX5 genomic lesions still need to be elucidated. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3624 Introduction: The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aim of our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods: the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results: The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions: in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Background: PTEN is a negative regulator of PI3K-AKT signaling and a potent tumor suppressor throughout all main types of human cancer. Recent work demonstrated that loss of PTEN in T cell acute lymphoblastic leukemia (ALL) confers glucocorticoid resistance. To develop a model for glucocorticoid resistance in pre-B ALL, we studied Cre-mediated deletion of Pten. Results: Surprisingly, loss of one or both alleles of Pten caused rapid cell death in pre-B ALL cells and was sufficient to cure transplant recipient mice from leukemia. Furthermore, deletion of Pten in pre-B cells disabled oncogenic transformation by BCR-ABL1 and NRASG12D oncogenes. A reanalysis of genetic lesions of PTEN in human cancer revealed a high frequency across all main subtypes of mature B cell lymphoma, myeloid leukemias and T cell lineage leukemia/lymphoma (11.3% in 1,697 samples). Strikingly, however, lesions of PTEN were not detected in any of 670 pre-B ALL patient samples. Whereas high expression levels of PTEN predict favorable outcomes for patients with T-ALL, pre-B ALL patients with high PTEN expression levels at the time of diagnosis have particularly poor clinical outcomes (P=0.01). Studying the unexpected role of PTEN in B cell lineage ALL, we developed a genetic model for Cre-mediated deletion of Pten in mouse pre-B ALL cells carrying BCR-ABL1 or NRASG12D oncogenes. Deletion of one or both alleles of Pten reduced the colony forming ability (p 〈 10E-06) and proliferation rate (p 〈 10E-04) of pre-B ALL cells while strongly increasing cellular senescence (P 〈 10E-03). Since immediate effects of Pten deletion include hyperactivation of PI3K-AKT signaling, we hypothesized that small molecule inhibitors that limit PI3K-AKT pathway activity may rescue the pre-B ALL cells from cell death when Pten is inducibly deleted. Therefore, we tested the effect of AKT and PI3K inhibitors AZD5363 and BKM120 on Pten deleted cells and confirmed that chemical inhibition of PI3K-AKT pathway indeed rescued cell death of pre-B ALL cells upon Pten deletion. Also, global tyrosine phosphoprofiling by quantitative mass spectrometry revealed that Cre-mediated deletion of Pten strongly induced activation of CBL, a ubiquitin ligase that induces degradation of the IL7 receptor in pre-B cells and oncogenic tyrosine kinases in leukemia. Genetic experiments confirmed that deletion of Pten and hyperactivation of AKT engaged a novel, CBL-dependent pathway of negative feedback inhibition of STAT5 and tyrosine kinase signaling. Consistent with these finding, Reverse Phase Protein Array (RPPA) measurements for 155 newly diagnosed cases of pre-B ALL revealed a strong negative correlation between STAT5 protein levels and phospho-ATKT308 (P=0.0021) and phospho-AKTS473 (P=0.0005). Unlike pre-B ALL, deletion of Pten in CML like cells showed no toxicity. To further study the lineage specifity of Pten dependency we used a doxycycline-inducible CEBPa construct to differentiate pre-B cell ALL into myeloid lineage leukemia cells. Similar to our finding in CML like cells, unlike the pre-B ALL, myeloid lineage Leukemia showed no dependency on Pten and the deletion of Ptencaused no significant change in the cells viability. Studying the basal level of PTEN, pre-B cells showed ~40-fold higher expression levels than myeloid lineage cells. Consistently, genetic knockdown of PTEN using two different shRNA was only toxic in patient-derived pre-B but not myeloid leukemia cells. Clinical relevance: To find out the clinical potential of our finding, we tested the effects of SF1670 a small molecule inhibitor of PTEN on a panel of pre-B ALL and CML. Interestingly, the pre-B ALL showed more sensitivity to PTEN inhibition compared to CML cells. Conclusion: Our analysis uncovered an unexpected vulnerability of human Pre-B ALL cells toward PTEN inhibition and hyperactivation of the PI3K-AKT pathway, which provides an explanation why activating mutations of this pathway are negatively selected in human pre-B ALL. Since short (few hours) and profound inhibition of Pten is sufficient to commit human pre-B ALL cells to cell death, transient inhibition of this pathway or transient hyperactivation of PI3K-AKT may be a promising strategy to target multi-drug resistant human ALL. Disclosures No relevant conflicts of interest to declare.