FIP200 controls the TBK1 activation threshold at SQSTM1/p62-positive condensates

The protein kinase TBK1 is a central regulator of innate immune responses and autophagy, and ablation of either function has been linked to neuroinflammatory or degenerative diseases. Autophagy is an intracellular process that recycles old or damaged proteins and organelles. In recent years, the TBK1-dependent regulation of autophagy pathways has been characterized. However, the autophagy-dependent regulation of TBK1 activity awaits further clarification. Here, we observed that TBK1 is recruited to SQSTM1/p62-containing aggregates via the selective autophagy receptor TAX1BP1. In these aggregates, TBK1 phosphorylates SQSTM1/p62 at serine 403 and thus presumably regulates the efficient engulfment and clearance of these structures. We found that TBK1 activation is strongly increased if FIP200, a component of the autophagy-inducing ULK1 complex, is not present or cannot bind to TAX1BP1. Given our collective findings, we hypothesize that FIP200 ensures the inducible activation of TBK1 at SQSTM1/p62 condensates.

Autonomous Ca2+ Oscillations Reflect Oncogenic Signaling in B-ALL Cells

Background: Engagement of the B-cell receptor (BCR) results in Ca2+ flux and is linked to B-cell survival based on store-operated Ca2+ entry (SOCE), which is triggered by ORAI1 and stromal interaction molecule-1 (STIM1). While normal B-cells release Ca2+ only in response to external stimuli (e.g. BCR-engagement), we recently observed that transformed B-cells exhibit autonomous oscillatory Ca2+ signals that are linked to oncogene activity. Studying a novel dual biosensor system to concurrently measure Ca2+ fluctuations and oncogenic kinase activity in the same B-ALL cells, we discovered that oncogenic kinase- and Ca2+-signals alternate between On- and Off-phases, during which kinase and Ca2+ signals are mutually exclusive. Results: Consistent with a scenario in which autonomous Ca2+-signaling is linked to oncogenic signaling, we observed that high mRNA levels of Orai1 and Stim1 in patients with B-ALL were linked to poor clinical outcomes. To study the role of SOCE and its effectors Orai1 and Stim1 in oncogene signaling, we developed mouse models for inducible deletion of Orai1 and Stim1/2 in BCR-ABL1- and NRASG12D-driven B-ALL. Tamoxifen-inducible activation of Cre in Orai1fl/fl and Stim1/2 fl/fl BCR-ABL1- and NRASG12D-driven B-ALL cells induced excision of both SOCE-mediators and induced near-complete loss of Ca2+-signaling competence. While treatment with the Ca2+ pump inhibitor, thapsigargin, elicited a strong SOCE signal, Ca2+-signaling was entirely muted upon Orai1-deletion, while deletion of Stim1 diminished and substantially delayed the residual Ca2+-signal. Studying autonomous Ca2+-signaling activity in B-ALL cells, Orai1-deletion resulted in a complete loss of oscillatory Ca2+-signaling, while deletion of Stim1/2 distorted the morphology of the Ca2+-signals with lowered amplitude and greatly extended signal duration. While both Orai1 and Stim1 contribute to SOCE, these observations highlight distinct roles of the two molecules in autonomous Ca2+ oscillation. For both BCR-ABL1- and NRASG12D-driven B-ALL, ablation of both Orai1- and Stim1/2 had profound effects on cell viability and the ability to form leukemic colonies. These results are consistent with the hypothesis that SOCE and autonomous Ca2+-oscillations are essential for oncogenic signaling in B-ALL and thus enable colony formation, proliferation and survival. To elucidate a functional link between autonomous Ca2+-oscillations and oncogenic signaling in B-ALL, we studied activation of Nfatc1, a central regulator of oncogene signaling and frequency-dependent decoder of Ca2+-oscillations. Upon Cre-mediated deletion of Orai1 and Stim1/2, however, Nfatc1 no longer translocated to the nucleus and was subject to increased proteasomal degradation. Establishing a functional link between Nfatc1-activation and survival and proliferation signaling in B-ALL cells, inducible activation of Nfatc1-deletion in BCR-ABL1-transformed Nfatc1flfl B-ALL cells phenocopied the effects observed with genetic deletion of Orai1 and Stim1/2. Interestingly, the NFATC1-inhibitor INCA6 exhibited strong cytotoxic responses in human B-ALL cells with strong Ca2+-oscillations, while Hodgkin's lymphoma and myeloma cells, lacking Ca2+-oscillations, were resistant to INCA6. To directly determine how oncogenic signaling impinges on Ca2+-oscillations, we developed a dual biosensor to concurrently measure Ca2+ fluctuations (RCaMP; red) and oncogenic BCR-ABL1 kinase activity (CRKL-phosphorylation; green) in the same B-ALL cells. Simultaneous measurement of Ca2+ (red) and kinase (green) activity revealed that B-ALL cells continuously alternated between Red-only and Green-only, suggesting that kinase and Ca2+ signals both oscillate in a mutually exclusive manner. Conclusions: Our findings show central roles of SOCE-effector genes, Orai1 and Stim1/2, in oncogenic signaling of B-ALL cells and activation of Nfatc1 as a driver of proliferation and survival signaling. Engineering of a dual biosensor for concurrent measurement of Ca2+ and oncogenic kinase activity revealed that B-ALL cells continuously alternate between Ca2+ and oncogenic kinase activity, with Ca2+-peaks marking the off-state of the oncogenic kinase. Disclosures No relevant conflicts of interest to declare.

Ras-Driven B-Cell Transformation Targets Developmental Rewiring of Cytokine to Pre-B Cell Receptor Signaling

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.