ALBERT

Description: Background: BCL6 is known as a protooncogene and transcriptional repressor in diffuse large B cell lymphoma, where it is frequently involved in chromosomal rearrangements. We recently identified BCL6 as a novel mediator of drug resistance to tyrosine kinase inhibitors (TKI) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). In addition,BCL6 directly competes with the tumor suppressor BACH2 for p53 promotor binding to protecting cells from p53-mediated apoptosis in multiple ALL subgroups (Swaminathan et al., Nature Medicine 2013). Based on this, our current study is focusing on the function of BCL6 in different subtypes of human ALL. Results: Analysis of gene expression data from 207 children with high-risk B cell precursor ALL (COG P9906) showed that high expression levels of BCL6 at the time of diagnosis correlated with a poor overall and relapse-free survival (p=0.007). Furthermore, 49 matched sample pairs from patients at diagnosis and relapse showed increased BCL6 levels at relapse compared to diagnosis (p=0.003). To test whether or not there are specific subtypes of leukemia with high BCL6 expression levels, we studied BCL6 expression via western blot and immunohistochemistry staining in Non-Ph+ cell lines and ALL patient samples (n=76). Interestingly, BCL6 levels are particularly elevated in MLL-rearranged (MLLr) ALL cases. In addition, patients from the clinical trial that had high BCL6 levels and had MLL rearrangements had the worst clinical outcome (p=0.0009). We next tested if MLLr oncogenes drive aberrant BCL6 expression. First, we performed a ChIP-analysis using antibodies against MLL, AF4, and ENL, which provided evidence for direct binding to the BCL6 promoter. We then performed a BCL6 Western blot analysis of inducible MLL-AF4-transgenic and retrovirally transduced MLL-ENL pre-B cells, demonstrating that both oncogenes are sufficient to induce ~10-fold upregulation of BCL6 protein levels. Additionally, we used a newly developed conditional BCL6 knock out/reporter mouse model to decipher the function of BCL6. We transduced B cell progenitor cells from BCL6fl/fl mice with MLL-ENL and either with a control or Cre-expression vector. Using the BCL6 reporter capability of the mouse we found that BCL6 is significantly higher expressed in MLL-ENL transduced cells. To test if MLL is required for BCL6 upregulation, we used a conditional MLL knock out mouse and found that after Cre-mediated deletion of MLL, pro-B, mature-B, and MLL-AF4 transduced ALL cells almost lost the ability to upregulate BCL6. Interestingly, using inducible BCL6 transgenic and knockout as well as retrovirally transduced pre-B and ALL cells showed that overexpression of BCL6 leads to higher expression levels of MLL and deletion of BCL6 results in lower expression levels of MLL. Therefore, we conclude that MLL and BCL6 both cooperate and activate each other's expression in an activating feedback loop. Strikingly, Cre-mediated BCL6- deficiency results in apoptosis of MLL-ENL transduced cells. Clinical relevance: To verify if the high BCL6 expression levels in MLL-AF4 patients are important for the disease progression, we transduced primary human ALL xenografts with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if pharmacological inhibition of BCL6 is of potential use for patients with MLLr leukemia, we treated multiple MLL-AF4 rearranged human xenograft cases with a RI-BPI a BCL6 peptide inhibitor. Strikingly, treatment with RI-BPI not only compromised colony formation in methylcellulose it also prevents leukemia-initiation in transplant recipient mice. RI-BPI also had a strong synergistic effect when combined with the chemotherapy drug Vincristine, which represents the backbone for most high risk regimen in pediatric ALL. Conclusions: These findings identify BCL6 as a central factor in leukemia initiation and survival and its pharmacological inhibition as a novel strategy to treat MLL-rearranged ALL. Aberrant expression of BCL6 in MLLr ALL is the direct consequence of the MLLr oncogenic activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors with currently used chemotherapeutics as potential approach to reduce the risk of ALL relapse and improve overall outcome. Disclosures Armstrong: Epizyme, Inc: Consultancy. Ernst:Amgen: Other: stocks. Melnick:Janssen: Research Funding.

Description: Introduction : Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase negatively regulates a diverse set of signaling pathways promoting tumor growth. While PP2A functions as tumor suppressor in multiple types of cancer, here, we demonstrate an unexpected pro-survival role of PP2A in pre-B acute lymphoblastic leukemia (ALL) cells. By calibrating PI3K-AKT-mTOR signaling strength, PP2A regulates glycolysis rate and thereby balances energy demands against anti-oxidant protection of pre-B ALL cells. In addition, PP2A reinstates activity of FOXO factors by dephosphorylation and thereby enables the anti-oxidant function of FOXO1 and FOXO3. Consistent with previous studies, we find PP2A is dispensable for the survival of myeloid leukemia cells which indicates a lineage-specific role of PP2A. Combined with genetic study and small molecule inhibitor, we verify that regulation in patient-derived xenografts and highlight PP2A as a therapeutic target in pre-B ALL. Results: Consistent with a divergent role of PP2A in pre-B ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. Consistent with these findings, mutations in PP2A subunits are extremely rare in B cell malignancies but relatively common in solid tumors and myeloid malignancies (COSMIC). We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A and the catalytic subunit C, which leads to near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of FoxO1, FoxO3a, p70S6K and S6 ribosomal protein, which indicated elevated PI3K-Akt-mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. PI3K and mTOR inhibitors also have rescue effect on PP2A deficient ALL cells in growth-competition assay. Luciferase-labeled PP2A-deleted ALL cells showed reduced cell growth and leukemia progression after being transplanted into recipient mice. However, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. This lineage-specific role of PP2A was verified by inducible CEBPα expression to reprogram B cell lineage ALL cells into myeloid cells. Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in Ph+ ALL but not in CML cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. By employing glucose flux metabolic profiling assay using [1,2-13C2]-D-glucose tracer, we found elevated glycolysis and repressed pentose phosphate pathway (PPP) flux in PP2A-deleted pre-B ALL cells. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted pre-B ALL cells, together with decreased anti-oxidant gene expression, increased DNA damage, including H2AX phosphorylation and p53 expression. The mechanistic role of ROS downstream of PP2A was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells. The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation. Conclusion: Here we revealed an unexpected role of PP2A in maintaining redox homeostasis in pre-B ALL cells. By regulating AKT-mTOR signaling, PP2A keeps the balance of glycolysis and PPP to meet the energy demands of pre-B ALL cells and avoids extreme levels of oxidative stress. We confirmed this pro-survival role of PP2A in both genetic mouse ALL model and human Ph+ ALL-patients derived leukemia cells. These findings highlight PP2A as a therapeutic target and suggest that agents like the PP2A inhibitor LB-100 may be of interest for pre-clinical development and testing. Disclosures No relevant conflicts of interest to declare.

Description: The Sox4 transcription factor mediates early B-cell differentiation. Compared with normal pre-B cells, SOX4 promoter regions in Ph+ ALL cells are significantly hypomethylated. Loss and gain-of-function experiments identified Sox4 as a critical activator of PI3K/AKT and MAPK signaling in ALL cells. ChIP experiments confirmed that SOX4 binds to and transcriptionally activates promoters of multiple components within the PI3K/AKT and MAPK signaling pathways. Cre-mediated deletion of Sox4 had little effect on normal pre-B cells but compromised proliferation and viability of leukemia cells, which was rescued by BCL2L1 and constitutively active AKT and p110 PI3K. Consistent with these findings, high levels of SOX4 expression in ALL cells at the time of diagnosis predicted poor outcome in a pediatric clinical trial (COG P9906). Collectively, these studies identify SOX4 as a central mediator of oncogenic PI3K/AKT and MAPK signaling in ALL.

Description: Background: The BCR-ABL1 tyrosine kinase in Ph+ ALL and other ABL1-kinase fusions and JAK-kinase lesions that are found in the emerging subset of Ph-like ALL cause malignant transformation through constitutive phosphorylation of STAT5 at Y694. In addition, genetic lesions of genes encoding IL7R, CRLF2, EPOR are frequently found in Ph-like ALL and cause constitutive STAT5 Y694-phosphorylation. Ph+ ALL represents the most frequent subtype of human ALL in adults and accounts for ~30% of adult B-lineage ALL cases. Ph-like ALL is frequent among children (~10%) and young adults (~25%). Both Ph+ and Ph-like ALL represent highly aggressive diseases and define the group of patients with the worst clinical outcome. STAT5 induces its own negative feedback through transcriptional activation of CISH and SOCS proteins. The cytokine inducible SH2-containing protein (CISH) binds to tyrosine kinase with its SH2 domain and induces its degradation through poly-ubiquitinylation and recruitment of cullins and other ubiquitin-ligases to its SOCS-box. Suppressor of cytokine signaling (SOCS) proteins can interfere with oncogenic tyrosine kinase signaling directly through its kinase inhibitory region (KIR). Interestingly, SOCS2, SOCS3 and CISH are highly expressed in Ph+ ALL and represent the focus of this study. Results: Gene expression analyses showed that mRNA levels of SOCS2 and CISH were 10-fold (p

Description: Background: The Ser/Thr Protein phosphatase 2A (PP2A) attenuates activity of RAS-ERK and PI3K-AKT signaling pathways and functions as important tumor suppressor in chronic myeloid leukemia (CML) by downregulating output of oncogenic BCR-ABL1 signaling. Restoration of PP2A activity has been proposed for the treatment of CML and eradication of TKI-resistant leukemia CML-stem cells (Neviani et al., 2013). While the tumor suppressor function of PP2A was independently confirmed by multiple groups, our analyses of PP2A function during early B cell development suggested a role for PP2A to contribute to B cell survival. Therefore, we studied whether PP2A has different roles in B-lineage ALL and CML and whether these mechanistic differences are relevant for patients with B-lineage and B-lymphoid blast crisis CML. Results: Consistent with a divergent role of PP2A in B-lineage ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A but also the catalytic subunit C and was paralleled by near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of p70S6K and S6 ribosomal protein, which indicated elevated mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. In agreement with previous studies, however, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. To verify that the observed difference between B cell and myeloid disease was indeed lineage-dependent, we reprogrammed B cell lineage ALL cells into myeloid CML using inducible overexpression of CEBPα. As expected, CEBPα-driven myeloid lineage conversion prevented cell death upon deletion of PP2A, demonstrating that PP2A-function represents a B cell-specific vulnerability. We next transplanted luciferase-labeled Ppp2r1afl/fl ALL cells into recipient mice and monitored cell growth and leukemia progression. Cre-mediated deletion significantly prolonged overall survival of recipient mice that were transplanted with Ppp2r1afl/fl ALL cells. Although mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles in all cases studied demonstrating that fatal leukemia arose from few subclones that had escaped Cre-mediated deletion of PP2A. Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in B cell lineage Ph+ ALL but not CML-like cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted ALL cells, together with decreased anti-oxidant gene expression, increased H2AX phosphorylation and p53 expression indicated impaired balance of glycolytic flux may account for increased death of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells. The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation. Conclusion: Cre-mediated ablation of PP2A in mouse Ph+ ALL cells induced rapid cell death through excessively oxidative stress but not in myeloid CML cells. We confirmed this pro-survival role of PP2A in human Ph+ ALL-patients derived leukemia cells through both CRISPR-Cas9 mediated genetic study and PP2A inhibition by a novel small molecule LB-100 (Jie Lu et al., 2009). These findings highlight PP2A as a therapeutic target with potential relevance in Ph+ ALL and B-lymphoid blast crisis progression of CML. Disclosures No relevant conflicts of interest to declare.

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: Background and Hypothesis: The transcriptional repressor and proto-oncogene BCL6 is a therapeutic target in subtypes of diffuse large B cell lymphoma (DLBCL) and modulates drug-resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL; Duy et al., Nature 2011). BCL6 was shown to be a critical factor that bypasses p53-dependent senescence and thereby enables RAS-driven transformation of mouse embryonic fibroblasts (Shvarts et al., Genes Dev. 2002). Given that ~50% of pediatric ALL cases carry genetic lesions that lead to hyperactivated RAS-ERK signaling (Zhang et la., Blood 2012), we examined the role of BCL6 in RAS-driven pre-B ALL and identified a novel mechanism by which RAS-ERK signaling can mediate BCL6 expression. Results: Using a doxycycline-inducible TetOn- NRASG12D vector system, we found that inducible activation of RAS-ERK signaling strongly upregulated BCL6 expression at both the mRNA (~350-fold) and protein (~50-fold) levels in murine pre-B cells. Increases in BCL6 expression were abrogated upon treatment with a MEK inhibitor (PD325901). In addition, Cre-mediated deletion of Mapk1 suppressed upregulation of BCL6 expression upon imatinib treatment in BCR-ABL1-driven pre-B ALL cells. These findings suggested that elevated expression of BCL6 is a consequence of ERK activation. Previously, we demonstrated that BCL6 expression is negatively regulated by STAT5 in BCR-ABL1 pre-B ALL (Duy et al., Nature 2011). Interestingly, oncogenic NRASG12D inhibited phosphorylation of STAT5-Y694 by activating the inhibitory protein tyrosine phosphatase Ptpn6. Cre-mediated deletion of Ptpn6 induced STAT5 activity. Furthermore, loss of Ptpn6 function abrogated upregulation of BCL6 expression induced by imatinib in BCR-ABL1 pre-B ALL. Taken together, RAS-ERK signaling induces BCL6 expression by suppressing STAT5 activity. To directly test the role of BCL6 in RAS-transformed pre-B ALL, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. Inducible deletion of Bcl6 in NRASG12D-transformed pre-B ALL cells led to rapid depletion from the cell culture and reduced colony forming ability in vitro. These findings suggested that BCL6 is required for maintenance of fully established RAS-transformed ALL. Notably, we found that initiation of NRASG12D-driven leukemia in vivo depends on BCL6 as NRASG12D ALL failed to give rise to leukemia in the absence of Bcl6 in transplant recipient mice. Studying a diagnostic (KRAS wild-type) and a relapsed (KRASG12V) sample from one pre-B ALL patient revealed increased BCL6 expression in KRASG12V relapsed ALL cells. In addition, selective sensitivity to PD325901 and a retro inverso BCL6 peptide inhibitor (RI-BPI) was observed in KRASG12V relapsed ALL cells. Finally, RI-BPI prolonged overall survival of recipient mice transplanted with KRASG12V relapsed ALL cells in vivo. Conclusions: In summary, we demonstrated a novel mechanism by which oncogenic RAS signaling induces expression of BCL6, and showed that BCL6 is critical for RAS-driven transformation in pre-B ALL. Importantly, ALL clones often acquire drug resistance and activating mutations in the RAS pathway (Bhojwani and Pui, Lancet Oncol. 2013). Our findings suggest that pharmacological inhibition of BCL6 may provide a novel therapeutic avenue to overcome drug-resistance and prevent leukemia relapse after initial remission in RAS-driven ALL. Disclosures Melnick: Janssen: Research Funding.