ALBERT

Description: Background B cells are selected at multiple developmental checkpoints for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: either insufficient or hyperactive signaling (e.g. from an autoreactive BCR) results in cell death. Acute lymphoblastic leukemia (ALL) is the most frequent type of cancer in children and typically arises from pre-B cells, a large fraction of which are autoreactive. In ∼25% of patients, ALL is driven by an oncogenic tyrosine kinase (e.g. BCR-ABL1 in Ph+ ALL) and defines the ALL subgroup with the worst clinical outcome. Ph+ ALL cells invariably develop resistance against tyrosine kinase inhibitors (TKI). Here we tested the hypothesis that inherent mechanisms of negative selection to eliminate autoreactive clones with hyperactive pre-BCR signaling are still active in transformed pre-B cells and identified a potential therapeutic target for ALL patients. Results The BCR-ABL1 oncogene mimics a constitutively active pre-BCR and an incremental increase of pre-BCR downstream signaling (ITAM overexpression) was indeed sufficient to induce cell death in Ph+ ALL, but not in normal pre-B cells with low baseline signaling strength. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. Patient-derived Ph+ ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1, CD300A and LAIR1. However, ITAM containing activation receptors like CD79B was absent on the cell surface, and there was point or frame-shift mutation for both CD79A and CD79B. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials. In the COG trial (P9906; n=207) for children high-risk ALL, mRNA levels of PECAM1, CD300A and LAIR1 at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p

Description: Key Points We evaluated interference with integrin alpha4–mediated stromal adhesion as a new acute lymphoblastic leukemia treatment. Integrin alpha4 blockade using natalizumab in combination with chemotherapy sensitizes pre-B acute lymphoblastic leukemia to chemotherapy.

Description: Abstract 513 Background: The phosphatase and tensin homolog (PTEN) tumor suppressor is a negative regulator of PI3K/AKT signaling and frequently deleted in solid tumors and in T cell lineage acute lymphoblastic leukemia (ALL). Recent work by our group demonstrated that Pten-deletion cooperates with WNT/β-catenin signaling in self-renewal signaling of leukemia stem cells in T cell lineage ALL (Guo et al., Nature 2008). Although PTEN deletions are uncommon in B cell lineage ALL and chronic myeloid leukemia, recent work identified Pten as a tumor suppressor in B cell lineage ALL and chronic myeloid leukemia (CML; Peng et al., Blood 2010). This study showed that Pten-deletion accelerated leukemia in B cell lineage ALL and CML and increased self-renewal capacity of leukemia stem cells. Approach: To investigate the mechanism of Pten-mediated negative regulation of leukemia proliferation and stem cell self-renewal in B cell lineage ALL and CML, we established a conditional mouse model for inducible ablation of Pten in BCR-ABL1-driven B cell lineage ALL and CML. To this end, B cell precursors or Lin− Sca-1+ c-Kit+ (LSK) cells from the bone marrow of Pten-fl/fl mice were transduced with retroviral BCR-ABL1 under B lymphoid or myeloid conditions. When growth-factor-independent B cell lineage or CML-like leukemia formed, Pten-fl/fl leukemia cells were transduced with tamoxifen (4-OHT)-inducible Cre-ERT2 or an ERT2 empty vector control. Results: Deletion of Pten was induced by 4-OHT treatment and near-complete deletion of Pten was observed on day 2 after Cre-ERT2 induction as determined by genomic PCR and Western blot. As expected, deletion of Pten resulted in a strong increase of phospho-AKT, as determined by Western blot. Surprisingly, deletion of Pten in B cell lineage leukemia and CML-like disease did not accelerate leukemia cell growth and had the opposite effect. After three days of Cre-induction, the vast majority of both B cell lineage and CML-like leukemia cells underwent cellular senescence, as measured by staining for senescence-associated β-galactosidase activity (〉100-fold increase; p=0.0008). In addition, Pten-deletion induced cell cycle arrest in both G0/G1 and G2/M phase of the cell cycle, which is consistent with cellular senescence. While growth kinetics and viability of Pten-fl/fl leukemia cells carrying the ERT2 empty vector control remained unchanged, Pten-deletion caused a reduction of viability by 80% within 6 days. Compared to B cell lineage leukemia and CML-like leukemia, Pten protein levels were very low in normal B cell precursors and myeloid progenitor cells and the consequences of Pten-deletion were less drastic in normal as compared to leukemia cells. Consistent with cellular senescence, deletion of Pten resulted in dramatic upregulation of p53 and p21 but not p27 cell cycle inhibitors. We recently identified BCL6 as a FoxO-dependent suppressor of p53 in BCR-ABL1-driven leukemias (Duy et al., J Exp Med 2010). Since Pten functions as a positive regulator of FoxO1, FoxO3A and FoxO4, we tested whether excessive upregulation of p53 and cellular senescence were a consequence of loss of BCL6/FoxO function downstream of Pten-deletion. Treatment of BCR-ABL1 B cell lineage and CML-like leukemia cells with Imatinib results in strong upregulation of BCL6 expression downstream of FoxO factors. Upon deletion of Pten, however, Imatinib-treatment failed to upregulate BCL6. In the absence of Pten, BCL6 protein expression was undetectable and p53 protein levels were excessively increased. Conclusion: Pten has been extensively studied as a tumor suppressor in a broad range of malignancies. It is frequently deleted in solid tumors and also in T cell lineage ALL, where Pten-deletion accelerates leukemia cell growth by increased PI3K/AKT survival signaling. In B cell lineage and CML-like leukemia, Pten deletion also increases PI3K/AKT signaling. Unlike T-ALL, however, B cell lineage leukemia and CML cells undergo cellular senescence and cell cycle arrest. In B cell lineage ALL and CML, the BCL6 transcriptional repressor is required to overcome p53-dependent senescence. Here we unexpectedly identify the PTEN phosphatase as a central requirement for FoxO-dependent upregulation of BCL6. Hence, PTEN signaling via FoxO/BCL6 is required for the ability of the BCR-ABL1 ALL and CML cells to evade p53-mediated cellular senescence. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2563 Background: SOCS (suppressors of cytokine signaling) are a family of intracellular proteins, including SOCS1–7 and CISH (cytokine induced SH2 domain protein). They play key roles in negative regulation of cytokine signal transduction, including JAK/STAT pathway. While BCR-ABL1 signaling involves STAT5 in Ph+ acute lymphoblastic leukemia (ALL) cells, STAT5 signaling also results in its own feedback inhibition via CISH, SOCS2 and SOCS3. Interestingly, CISH, SOCS2 and SOCS3 are highly expressed in Ph+ALL and represent the focus of this study. Results: Comparing microarray data for human Ph+ ALL (n=15) and normal human pre-B cells (n=8), we found that mRNA levels of SOCS2 and CISH were 10-fold (p

Description: Abstract 291 Background: ITIM (immunoreceptor tyrosine-based inhibition motifs) motifs in the cytoplasmic tail of inhibitory receptors recruit inhibitory phosphatases. Upon ligation, these phosphatases inhibit signal transduction from tyrosine kinases including BCR-ABL1. ITIM-receptors are critical in the control of immune responses of B and T cells. Surprisingly, we found that a number of inhibitory ITIM-receptors are expressed at very high levels on the surface of patient-derived Ph+ acute lymphoblastic leukemia (ALL) cells compared to normal pre-B cells. In this study, three central ITIM-receptors were identified and analyzed for their function in Ph+ALL: PECAM1 (platelet/endothelial cell adhesion molecule 1/CD31), CD300A and LAIR1 (leukocyte-associated immunoglobulin-like receptor 1). Results: Microarray data showed mRNA levels of PECAM1, CD300A and LAIR1 were about 5-fold higher (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: 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.

Description: Background & Hypothesis: The Philadelphia chromosome encoding the oncogenic BCR-ABL1 (Ph+) tyrosine kinase represents a common transforming oncogene in both chronic myeloid leukemia (CML) and a subset of pre-B acute lymphoblastic leukemia (ALL). While both diseases are driven by the same oncogene, biological and clinical characteristics differ between CML and Ph+ ALL. Given that oncogenic signaling from potent tyrosine kinases like BCR-ABL1 imposes significant metabolic requirements on energy supply, biogenesis and ability to survive metabolic stress, we investigated whether the divergent characteristics of BCR-ABL1 CML and Ph+ ALL have a metabolic basis. Results: Interestingly, metabolic analyses demonstrated that patient-derived CML chronic phase (CML-CP) and Ph+ ALL cells have distinguishable glycolytic profiles. Consistent with a higher proliferative rate, Ph+ ALL cells rapidly exhausted the capacity of their glycolytic machinery when compared to CML-CP cells. Furthermore, when comparing their glycolytic reserve (i.e. spare glycolytic capacity needed to compensate for loss of mitochondrial ATP production), Ph+ ALL cells were shown to have a lower glycolytic reserve compared to CML-CP cells. These findings suggest that Ph+ ALL cells have less flexibility in adapting to metabolic stress. Searching for factors that mediate metabolic reprogramming to support rapid cellular proliferation and promote adaptation to metabolic stress in a lineage-specific manner, we identified the tumor suppressor LKB1 and its substrate AMPK which coordinate metabolism with cell growth. To elucidate the potential lineage-specific functions of LKB1, genetic mouse models for 4-hydroxytamoxifen (4-OHT)-inducible deletion of Lkb1 in BCR-ABL1-driven myeloid leukemia (CML-like) and pre-B ALL (Ph+ ALL-like) were developed. Cre-mediated deletion of Lkb1 in myeloid leukemia mediated proliferation and stimulated aerobic glycolysis, as reflected by increases in both glucose consumption and lactate production. These findings are in agreement with previous findings in solid tumors and are consistent with the role of LKB1 as a tumor suppressor. In striking contrast, deletion of Lkb1 in pre-B ALL cells induced apoptosis and cell cycle arrest. In vivo, Lkb1 deletion in pre-B ALL cells delayed leukemia initiation and prolonged overall survival of transplant recipient mice. Furthermore, bioenergetics measurements of glycolytic and mitochondrial functions in response to various conditions of metabolic stress revealed that loss of Lkb1 function in pre-B ALL resulted in impaired metabolic adaptation. Likewise, metabolite profiling revealed that deletion of Lkb1 in pre-B ALL cells resulted in altered levels of metabolites from glycolysis, the TCA cycle, the pentose phosphate pathway, nucleotide metabolism, currency metabolites, amino acid metabolism and fatty acid metabolism. Importantly, C/EBPα-mediated lineage reprogramming of pre-B ALL cells into the myeloid lineage alleviated their dependency on Lkb1. Similar to observations made with Cre-mediated deletion of Lkb1 in a model for BCR-ABL1 pre-B ALL, small molecule inhibition of AMPK using BML-275 (an ATP-competitive inhibitor) induced apoptosis in patient-derived pre-B ALL cells. Moreover, BML-275 had deleterious effects on metabolic adaptation in patient-derived pre-B ALL cells, but not in CML-CP cells. Finally, BML-275 synergized with pharmacological inhibition of PI3K/Akt signaling in inhibiting glycolytic function and eradicating patient-derived pre-B ALL cells. Combining BML-275 and a PI3K inhibitor (BKM120) exerted significantly more potent inhibitory effect on pre-B ALL progression than each agent alone, prolonging the overall survival of recipient mice. Conclusions: Taken together, our findings demonstrate that LKB1/AMPK signaling plays lineage-specific roles in BCR-ABL1-driven CML and pre-B ALL. While LKB1/AMPK is an established tumor suppressor pathway in multiple cell types, our findings reveal a unique and previously unrecognized vulnerability of pre-B ALL cells. Disclosures No relevant conflicts of interest to declare.