ALBERT

Beschreibung: Background and rationale. Leukemia-initiating cells (LIC) were extensively studied in AML (Bonnet 1997) and CML (Graham 2002), while LIC populations in B-ALL remained elusive. In the absence of a functional definition, targeted approaches for LIC-eradication are not feasible in B-ALL. Some studies suggested immunophenotypes for B-ALL LIC-populations (Cox 2004, Castor 2005, Wang 2007). However, other groups demonstrated that tumor-initiation in B-ALL is not limited to rare populations or developmental hierarchy (Kelly 2007; Le Viseur 2008; Rehe 2013; Aoki 2015). Concept: We hypothesize that self-renewal in the B-cell lineage is induced by positive selection and antigen-receptor (BCR) signaling, i.e. encounter of cognate antigen. Self-renewal at this stage leads to clonal expansion and survival. Unlike stemness in AML and CML, which is determined by a developmental hierarchy, we propose that self-renewal in the B-cell lineage is transient and driven by environmental antigen and the ability of BCRs to bind with high affinity. In B-cell malignancies, positive B-cell selection events, resulting in Lgr5 surface expression, are mimicked by transforming oncogenes (e.g. BCR-ABL1, NRASG12D, MYD88L265P). Results: Combining flow cytometry and genetic approaches, we identified surface expression of the leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) as new biomarker of positively selected pre-B cells in the bone marrow. Conversely, conditional ablation of Lgr5 during earliest stages of B-cell development resulted in near-complete failure to develop a mature B cell pool (reduced by 2-3 log orders). Lgr5 is a Wnt target gene and an established cancer stem cell marker for epithelial cancers (e.g. colorectal cancer and mammary tumors), however, a role for Lgr5 in normal and malignant hematopoiesis is not known. Importantly, Lgr5 represents a previously unrecognized predictor of poor clinical outcome in children and adults with pre-B ALL, including worse overall survival and higher risks of drug-resistance and relapse. Limiting dilution transplant experiments showed that Lgr5-overexpression increased LIC-frequencies in NSG recipient mice. Inducible activation of Cre in Lgr5fl/fl mouse models for BCR-ABL1- or NRASG12D-driven B-ALL resulted in cell cycle arrest, abolished colony forming capacity and compromised the ability of leukemia cells to initiate fatal disease in NSG transplant recipients. Deletion of Lgr5 in pre-B ALL cells caused massive accumulation of nuclear β-catenin and increased expression of β-catenin target genes. Phosphoproteomic analyses revealed increased levels of β-catenin S675-phosphorylation, which increases β-catenin transcriptional activity (Taurin 2006; Hino 2015). Inducible activation of a gain-of-function mutant of β-catenin revealed that pre-B ALL cells are extremely sensitive to β-catenin activation. Thus, Lgr5 enables positive selection and self-renewal of B-ALL cells by curbing β-catenin activity. Therapeutic implication: To assess Lgr5 surface expression on B-ALL as a target for antibody-drug conjugate (ADC), we treated refractory B-ALL PDX with the Lgr5-MMAE ADC. Single-agent treatment with Lgr5-MMAE significantly reduced B-ALL leukemia burden. Treatment with dexamethasone not only enforced persistent surface expression of Lgr5, but also potentiated efficacy of by Lgr5-MMAE. Conclusion: Unlike self-renewal in myeloid leukemia that is determined by a developmental hierarchy, our results here show that self-renewal in the B-cell lineage is transient and driven by the ability of BCRs to bind antigen with high affinity. Lgr5 is a biomarker of this selection event, critical for the initiation of B-ALL and other B-cell malignancies in transplant recipients. Given that positive B-cell selection events, resulting in Lgr5 surface expression are mimicked by transforming oncogenes, Lgr5 also represents a promising target for ADC therapy for instance Lgr5-MMAE (Genentech). Disclosures No relevant conflicts of interest to declare.

Beschreibung: Studying gene expression and clinical outcome data from 136 clinical trials for patients with cancer (~21,000 patients with 26 cancer types), we found CD25 as one of the strongest predictors of poor clinical outcome in patients with B-cell malignancies, but not in other cancer types. This was unexpected because CD25 is known as one of three chains of the IL2 receptor on T-cells and NK-cells. Interleukin-2 (IL2) functions as essential T-cell growth factor. IL2 signals through β- and γ-, but not α-chains (CD25) of its heterotrimeric receptor. CD25-deficiency causes lymphoproliferation and autoimmunity, however, its mechanistic role is unclear. Our experiments based on genetic mouse models and engineered patient-derived B-cell leukemia and lymphoma xenografts revealed that CD25 expressed on B-cells is not an IL2 receptor chain, but in fact binds the B-cell receptor (BCR) to regulate its activity. Suggesting IL2-independent functions, defects in CD25-/-B-cells were not replicated in IL2-deficient mice. CD25 bound the BCR but not IL2Rβ- and IL2Rγ-chains. IL2Rβ- and IL2Rγ-chains can pair with other chains to form receptors for different cytokine-ligands. However, CD25 represents the first example of a cytokine receptor chain that binds to the BCR for negative feedback regulation. Likewise, in T-cells, CD25 had a bifunctional role and either functioned as IL2 receptor chain or as negative feedback regulator of T-cell receptor signaling. CD25-function was regulated by cell-membrane translocation, which required phosphorylation of its cytoplasmic tail at S268 (see schematic, left). In a family with monogenic autoimmunity, a mutation immediately preceding S268 compromised CD25-surface translocation, which was restored by homology-directed repair of the S268 motif. CD25-interactome analyses identified PKCd as critical effector molecule downstream of CD25 to mediate B-cell selection during normal B-cell development and calibrate oncogenic BCR signaling in B-cell tumors. In B-cell malignancies, BCR-dependent survival and proliferation signals are often substituted by oncogenic BCR-mimics (e.g. BCR-ABL1, JAK2, BRAFV600E, LMP2A, CD79B mutations; see schematic, right). Accordingly, we identified CD25 surface-expression as biomarker of oncogenic BCR-signaling and predictor of poor clinical outcomes. CD25-/-B-cell leukemia failed to initiate fatal disease in transplant recipients. Owing to imbalances of oncogenic BCR-signaling and p53-checkpoint activation, CD25-/- B-cell leukemia failed to initiate fatal disease in transplant recipients. In patient-derived xenograft models of drug-resistant B-cell malignancies, treatment with a CD25-specific antibody drug-conjugate (ADCT-301) extended survival of transplant recipients or eradicated disease. These findings identified CD25 as previously unrecognized feedback regulator of oncogenic BCR-signaling and provide a rationale for therapeutic targeting of CD25 in refractory B-cell malignancies. Figure. Figure. Disclosures Forman: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Weinstock:Genentech/Roche, Monsanto: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Travera: Equity Ownership; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties. Uzel:Novartis: Research Funding.

Beschreibung: Background & Hypothesis: B cell receptor (BCR) signaling and oncogenic tyrosine kinases that mimic BCR-signaling in B-lineage leukemia and lymphoma depend on assembly of membrane proximal signaling complexes. Signalosomes in normal BCR- and oncogene (e.g. BCR-ABL1, RAS-pathway lesions) signal transduction are recruited to phospholipid anchors in lipid rafts. The robustness of these complexes depends on cholesterol accumulation in lipid rafts. Here we identified the interferon-induced transmembrane protein IFITM3 as a central regulator of cholesterol in lipid rafts. Results: IFITM3 is mostly localized to endosomal compartments. By antagonizing VAP-A and oxysterol-binding protein 1 (OSBP1), IFITM3 promotes cholesterol accumulation and solidifies the endosomal membrane. This mechanism is particular important in anti-viral immunity, to "trap" intraluminal viral particles for lysosomal degradation. In B-cells, IFITM3 can translocate to the cell membrane and form a complex with the BCR and its co-receptors CD19, CD81 and CD21. While the functional significance of membrane expression of IFITM3 on B-cells was not known, we found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.006; n=207) and adults (ECOG E2993; P=0.014; n=215) with B-ALL. In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of relapse and positive MRD status at the end of induction chemotherapy in B-ALL and other B-cell malignancies. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B- ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion. While its membrane-topology can vary in different cell types, we found that IFITM3 functions as a dual-pass transmembrane protein in tight association with CD19 and the Iga and Igb signaling chains of the BCR in B-ALL and B-cell lymphoma cells. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRASG12D. Strikingly, deletion of IFITM3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRASG12D and failed to initiate fatal leukemia in transplant recipient mice. These changes were paralleled by G0/1 cell cycle arrest (P

Beschreibung: Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) was identified as interferon-inducible molecule in the context of viral infection. Endosomal membrane localized IFITM3 appear to prevent fusion events of intraluminal viral particles to the endosomal membrane through accumulation of cholesterol which makes membrane more rigid. We recently found that IFITM3 is a dual-pass transmembrane protein expressed on B cell lineage ALL cells. Thereby IFITM3 is associated with known B cell co-receptors including CD19, CD81 and CD21. While the significance of this association was unknown, we found that IFITM3 is required for surface expression of the B cell antigen CD19. Although immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013), in some cases, CD19-specific engineered chimeric antigen receptors (CART19) treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Results: Studying IFITM3 mRNA levels in B cell lineage ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of ALL relapse and positive MRD status at the end of induction chemotherapy. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRAS. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K signaling in both normal and malignant B cells. These changes were paralleled by G0/1 cell cycle arrest (P

Beschreibung: Background: IGLL1 and VPREB1 encode for the l5 and VpreB components, respectively, of the surrogate light chain (SLC) of the pre-B cell receptor (pre-BCR). During early B-cell development, immunoglobulin (Ig) heavy chains pairs with SLCs to form the pre-BCR, a central signaling unit that drives proliferation and survival. Accordingly, germline mutations of IGLL1 (Minegishi J Exp Med 1998) and VPREB1 (Conley Immunol Rev 2005) are associated with profound B-cell defects and agammaglobulinemia in humans. However, somatic deletions of VPREB1 gene are frequent lesions in B-ALL and occur in 〉10% of B-ALL cases (Mangum et al., Leukemia 2014; Geng et al., Cancer Cell 2015), the significance of which is not clear. Since VPREB1 deletions are typically present at the time of diagnosis and are rarely acquired as secondary lesions at the time of relapse (Kuster Blood 2011), we hypothesized that loss of VPREB1 represents an early and essential event in leukemogenesis. Experimental Approach and Results: For genetic gain and loss of function analysis of VPREB1 and IGLL1, we established leukemia models based on pre-B cells from Igll1-/- mice (Kitamura Cell 1992), and Vpreb1-Igll1 double-transgenic mice (Van Loo Immunity 2007). Loss of Igll1 completely abrogated SLC expression on the surface of pre-B cells. In contrast, VpreB1-Igll1 double-transgenic pre-B cells expressed constitutively higher surface levels of SLC as part of their pre-BCR as evidenced by flow cytometry. Compared to wildtype controls, Igll1-/- pre-B cells lacking the ability to express a functional SLCs were more readily transformed by BCR-ABL1 oncogene. However, pre-B cells of VpreB1-Igll1 transgenic mice, were not permissive to BCR-ABL1 mediated transformation. In agreement with these results, VpreB1-Igll1 double-transgenic pre-B cells were resistant transformation by BCR-ABL1 in vivo. BCR-ABL1-transgenic mice with enforced expression Vpreb1-Igll1 remained disease-free for more than nine months, whereas the vast majority of BCR-ABL1-transgenic mice downregulated pre-BCR surface expression and developed lethal B-ALL within 90 days of birth (n=34, P

Beschreibung: Background and significance: Activation of STAT5- and ERK-signaling are segregated to early and later stages of normal B cell development, respectively. While pro-B cells depend on activation of Stat5 downstream of cytokine receptors, pre-B and immature B cells depend on Erk-activation downstream of pre-B cell receptor (pre-BCR). Since Stat5- (cytokine receptor) and Erk- (pre-BCR) signaling antagonize each other, we studied evolutionary trajectories of Stat5- and Erk-activating lesions in B-cell lineage ALL. We tested the hypothesis that an initial activating mutation in one of the two pathways represents a commitment step that makes the tumor clone non-permissive to activating lesions in the other pathway. Results: Studying B-lineage ALL (n=578), we found that STAT5-activating (e.g. BCR-ABL1, JAK2, cytokine receptors) and ERK-activating (NRAS, KRAS, PTPN11, NF1) lesions were mutually exclusive, with only 9 cases (1.6%) carrying lesions in both pathways. Even in the few remaining cases, low allele frequencies of one of the two lesions suggests that they do not co-occur in the same cells. Reverse phase protein array measurements revealed that phosphorylation of ERK and its upstream kinase MEK were inversely correlated with STAT5-phosphorylation (MDACC, 1983-2007; P

Beschreibung: The spleen tyrosine kinase (SYK) and ζ-associated protein of 70 kD (ZAP70) tyrosine kinases play critical roles in proximal signal transduction of B-cell (BCR) and T-cell receptors (TCR), respectively. The highly similar SYK and ZAP70 kinases share a common structure composed of two tandem SH2 domains and a carboxy-terminal kinase domain. A linker region, termed interdomain B, connects the SH2 domains to the kinase domain and is important for kinase activation. Despite their conserved structure, SYK and ZAP70 are expressed in a largely mutually exclusive manner and play analogous roles in BCR- and TCR-signaling. Cross-lineage activation of ZAP70 in B cells was previously identified in chronic lymphocytic leukemia (CLL), which is characterized by clonal accumulation of malignant CD5+ B-cell cells that retain dependency on the BCR for survival signals. Nearly half of CLL cases show co-expression of SYK and ZAP70, and these patients have an aggressive disease course and a poor prognosis. Our analysis shows that in addition to CLL, aberrant ZAP70 expression occurs in other B-cell malignancies, e.g. TCF3-PBX1 pre-B ALL and B-cell lymphoma subsets that depend on survival signals from a functional (pre-) BCR. These findings suggest that interactions between SYK and ZAP70 may function to fine-tune strength of oncogenic BCR-signaling. To test this hypothesis, we have used a combination of molecular and proteomic approaches. We studied mechanisms by which ZAP70 integrates into BCR-mediated signals, and how the function of ZAP70 in B-cells differs from its native role downstream of the TCR. We demonstrate that ectopically expressed SYK and ZAP70 proteins are constitutively phosphorylated in BCR-ABL1+ B-ALL cells, but these induce distinctive signaling thresholds. CRISPR-mediated deletion of SYK or ZAP70 in leukemic cells further revealed that SYK and ZAP70 regulate unique signaling pathways in B-cells. We also demonstrate that ZAP70 is activated following BCR stimulation of lymphoma cells, and SYK/ZAP70 co-expressing cells display enhanced BCR signaling. Interestingly, enhanced BCR signaling was also observed in cells engineered to express an alternative splice variant of SYK (SYK-S). This shorter isoform of SYK, lacks a 23 amino-acid insert in the interdomain-B linker region, which is also absent in ZAP70, and may define unique protein-interactions that modulate signaling outcome. To elucidate the differential interactome of SYK, SYK-S, and ZAP70 we performed proximity-dependent biotin identification (BioID) experiments in B-cells following BCR-activation to capture the core signalling networks of these kinases in leukemic cells. In addition to expected BCR components including BLNK, PTPN6 and CBL we identified novel SYK and ZAP70 associated molecules including IKZF3, LAT2 and WAS which may play important roles in the survival of BCR-dependent malignancies. Importantly our findings highlight a role for ZAP70 in oncogenic BCR-signaling and suggest that ZAP70 promotes oncogenic BCR-signaling by limiting the ability of the BCR to induce negative B-cell selection and cell death. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Ras-pathway lesions are oncogenic drivers in ~45% of B-cell acute lymphoblastic leukemia (B-ALL) cases. Activating mutations of NRAS and KRAS are oncogenic drivers in B-ALL while the BRAFV600E mutation occurs in almost all cases of B-cell hairy cell leukemia. Less frequent lesions resulting in increased ERK-signaling in B-cell malignancies include activating mutations of RAF1, MAP2K1 and the PTPN11 phosphatase as well as deleterious mutations of the Ras-GTPase activator NF1. Interestingly, increased immunoglobulin light chain gene expression was observed in B-ALL cases with RAS-pathway lesions (COG P9906), reflecting engagement of pre-B cell receptor (pre-BCR) downstream signaling. Here we tested the hypothesis that oncogenic RAS-signaling in B-ALL mimics pre-BCR-induced developmental rewiring of signal transduction at the pro-B to pre-B cell transition and identified PTPN6 and BCL6 as therapeutic targets in RAS-driven B-ALL. During early B-cell development, pro-B cells transition from cytokine- to pre-B cell receptor (pre-BCR)-dependent survival and proliferation signals. Inducible activation of immunoglobulin (Ig) µ heavy chain (µHC) expression induced developmental progression and surface expression of Ig κ light chains. Notably, inducible activation of oncogenic NRASG12D had the same effect and resulted in increased surface expression of Ig κ light chains. Furthermore, studying genetic models for this transition revealed that both pre-BCR signaling and RAS-oncogenes suppressed cytokine receptor/STAT5-signaling and induced massive de novo expression of the proto-oncogene and transcriptional repressor BCL6. Our genetic studies revealed that the SH2-domain containing protein tyrosine phosphatase PTPN6 was activated by oncogenic RAS-signaling and essential for the switch from STAT5 to BCL6-activation. Given that oncogenic RAS activated PTPN6, we tested the role of PTPN6 in RAS-driven leukemogenesis. To this end, ablation of Ptpn6 in NRASG12D-driven B-ALL resulted in depletion of cells from cell culture in competitive-growth assays and reduced the number of colonies formed in semi-solid methylcellulose. Collectively, these findings suggest that PTPN6 represents a potential therapeutic intervention point in RAS-driven B-ALL. In addition to PTPN6, we investigated the role of BCL6 in RAS-driven B-ALL. Aberrant activation of oncogenic RAS results in oncogene-induced senescence (OIS) characterized by induction of ARF/p53 and irreversible cell cycle arrest in the G1 phase. For oncogenic Ras-signaling, BCL6 was required to oppose ERK-mediated activation of p21, p27 and p53 checkpoint molecules in B-ALL. Here we tested the hypothesis that BCL6 bypasses the RAS-mediated OIS program to facilitate transformation. To this end, increases in number of colonies formed in semi-solid methylcellulose were observed upon inducible activation of Bcl6 in NRASG12D B-ALL cells. Furthermore, loss of Bcl6 function in NRASG12D B-ALL cells resulted in depletion of cells from cell culture in competitive growth assays and reduced colony forming ability. Importantly, expression of NRASG12D in Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In striking contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in vivo. Furthermore, pharmacological inhibition of BCL6 restored sensitivity to chemotherapy in patient-derived KRASG12V B-ALL cells. In conclusion, we identified oncogenic RAS-signaling as functional mimics of pre-BCR signaling. Oncogenic RAS induced expression of BCL6 at the expense of cytokine receptor/STAT5-signaling. Our genetic studies identified PTPN6 as a critical effector molecule of the switch from cytokine receptor to pre-BCR signaling. Importantly, we identified PTPN6 and BCL6 as potential therapeutic intervention points in RAS-driven B-ALL. Figure. Figure. Disclosures No relevant conflicts of interest to declare.