scholarly journals Erk and Stat5 Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 787-787
Author(s):  
Shojaee Seyedmehdi ◽  
Zhengshan Chen ◽  
Maike Buchner ◽  
Christian Hurtz ◽  
Huimin Geng ◽  
...  
Keyword(s):  
B Cells ◽  
Feedback Control ◽  
B Cell ◽  
Small Molecule ◽  
Negative Feedback ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Targets ◽  
Feedback Regulation ◽  
Oncogenic Signaling ◽  
Negative Feedback Regulation

Abstract Background and Hypothesis: Targeted therapy of cancer typically focuses on the development of agents that will inactivate a transforming oncogene. In this study, we tested the concept that besides the oncogene itself, factors that enable permissiveness of a normal cell to oncogenic signaling represent a novel class of therapeutic targets. This hypothesis was based on three findings. First, acute activation of oncogenes in normal pre-B cells typically caused immediate cell death, unless pre-B cells were capable of adapting quickly enough to a high level of signaling output. Second, few surviving pre-B cell clones achieved permissiveness to oncogenic signaling by strong activation of negative feedback control of Erk and Stat5. Third, robust feedback control of Erk and Stat5 distinguishes normal pre-B cells from fully transformed pre-B acute lymphoblastic leukemia (ALL) cells. Results: To test the significance of strong feedback control of Erk and Stat5 signaling in pre-B ALL cells, we developed genetic loss-of function models for six central molecules in Erk (DUSP6, SPRY2, ETV5) and Stat5 (Cish, SOCS2, SOCS3) feedback control. Genetic deletion of the sprouty family Ras inhibitor Spry2, the Erk dual specificity phosphatase Dusp6 and their transcriptional activator Etv5, decreased robustness of Erk feedback control and compromised oncogenic transformation in mouse models for pre-B ALL. Likewise, ablation of Stat5 feedback control through deletion of the suppressors of cytokine signaling (SOCS) family molecules Cish, Socs2 and Socs3 reversed permissiveness of pre-B cells. Studying deletion of Spry2 (Erk) and Cish (Stat5) in an in vivo transplant model using inducible, Cre-mediated deletion of Spry2 and Cish in pre-B ALL cells confirmed that Erk and Stat5 feedback control are essential for malignant transformation and development of lethal leukemia. Genetic deletion of Erk (Dusp6, Spry2, Etv5) and Stat5 (Cish, Socs2, Socs3) feedback control impairs leukemic transformation of pre-B cells. Searching for factors that restrict permissiveness to oncogene signaling, we identified the pre-B cell tumor suppressor IKZF1, which is deleted in a large fraction of pre-B ALL cases. IKZF1 directly bound to and transcriptionally repressed multiple promoters of Erk and Stat5 feedback control and IKZF1 deletion raised the limit of maximum allowable oncogene signaling strength in pre-B ALL cells. We propose that the pre-B cell tumor suppressor IKZF1 functions as transcriptional repressor of Erk and Stat5 feedback control and thereby retains pre-B cells in a Non-permissive state. Clinical relevance: To assess potential usefulness of this finding for the development of future treatment strategies, we tested the effect of a specific small molecule inhibitor of DUSP6, E-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), which was designed as an allosteric inhibitor of the interaction between DUSP6 and phospho-ERK1/2. Interestingly, BCI acutely subverted Erk feedback control and selectively induced cell death in pre-B ALL cells. Small molecule inhibition of DUSP6 was sufficient to overcome conventional mechanisms of drug-resistance in pre-B ALL and selectively killed patient-derived pre-B ALL cells in a leukemia transplant model. BCI treatment, similar to Dusp6-deletion in our leukemia mouse model, led to the accumulation of P53 and ARF in patient-derived pre-B ALL cells. In addition, small molecule inhibition of DUSP6 had strong selective activity on drug-resistant patient-derived pre-B ALL cells that were injected into NOD/SCID transplant recipient mice. These findings identify permissive negative feedback control of oncogenic signaling as a previously unrecognized vulnerability of pre-B ALL cells and a new class of potential therapeutic targets. Conclusion: Targeting negative feedback regulation of both Erk and Stat5 signaling for the treatment of pre-B ALL seems counter-intuitive because it represents effectively the opposite of current efforts of targeted inhibition of oncogenic signaling. Our results, however, demonstrate that a robust negative feedback regulation is required for the leukemic transformation and development of fatal leukemia in pre-B ALL. We demonstrate that feedback control of Erk and Stat5 signaling represents a previously unrecognized vulnerability and, potentially, a novel class of therapeutic targets in human pre-B ALL. Disclosures No relevant conflicts of interest to declare.

Harnessing Negative B Cell Selection to Overcome Drug-Resistance in Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 792-792
Author(s):  
Zhengshan Chen ◽  
Seyedmehdi Shojaee ◽  
Huimin Geng ◽  
Jae-Woong Lee ◽  
Maike Buchner ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
B Cell ◽  
Negative Selection ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Targets ◽  
Inhibitory Receptors ◽  
Oncogenic Signaling ◽  
Bcr Signaling ◽  
Tki Treatment

Abstract Background: B cells are selected for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: attenuation below minimum (e.g. non-functional BCR) or hyperactivation above maximum (e.g. autoreactive BCR) thresholds of signaling strength causes negative selection and cell death. About 25% of B cell acute lymphoblastic leukemia (ALL) is driven by oncogenic tyrosine kinases (e.g. BCR-ABL1 in Ph+ ALL), which mimics constitutively active pre-BCR signaling and defines the ALL subgroup with the worst clinical outcome. Currently more potent tyrosine kinase inhibitors (TKI) are developed for Ph+ ALL to suppress oncogenic signaling below a minimum threshold for survival. However Ph+ ALL cells invariably develop resistance against TKI. Here, we tested the hypothesis that targeted hyperactivation of oncogenic signaling above a maximum threshold will trigger B cell-inherent mechanisms of negative selection and selectively kill Ph+ALL cells. Results: The Ph+ ALL cells don not express a functional pre-BCR and BCR-ABL1 oncogene mimics a constitutively active pre-BCR by phosphorylating SYK, LYN, BTK and PLCg2. An incremental increase of pre-BCR downstream signaling (ITAM or SYK overexpression) was indeed sufficient to induce cell death in Ph+ ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ALL cells in this experimental setting. Ph + ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1 (CD31), CD300A and LAIR1. 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 LAIR1at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p<0.0005), and negatively correlated with overall survival (OS) rate (p<0.02) or relapse free survival (RFS) rate (p<0.05). In the ECOG trial (E2993; n=215) for adults ALL, PECAM1 mRNA level negatively correlated with OS rate (p=0.0285). Genetic studies revealed that Pecam1, Cd300a and Lair1 receptors are critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases Ptpn6 (SHP1) and Inpp5d (SHIP1). Deletion of Pecam1, Cd300a or Lair1 in Ph+ ALL cells caused increased ROS levels, G0/G1cell cycle arrest, decreased colony formation capacity and cellular senescence. Phosphorylation of pre-BCR downstream molecules (SYK, LYN, BTK and PLCg2) was increased after Lair1 deletion and this hyper-signaling could not be tolerated by Ph+ ALL cells. Lair1 deletion resulted in rapid leukemia regression and prolonged survival of recipient mice in a transplant experiment. Leukemia cell death caused by Lair1-deletion could be rescued by overexpression of the active inhibitory phosphatase Ptpn6 (CD8-SHP1) or Inpp5d (CD8-SHIP1). Genetic deletion of Ptpn6 and Inpp5d caused increased pre-BCR signaling and cell death in BCR-ABL1 ALL cells but not myeloid cells (normal and BCR-ABL1-transformed), which -unlike B cells- are not subject to negative selection of auto-reactive clones. Decreasing pre-BCR signaling by SYK inhibition rescued cell death after Ptpn6- or Inpp5d- deletion. Blocking inhibitory receptors by using chimeric PECAM1, CD300A and LAIR1 receptor decoys inhibited proliferation and caused cell death in Ph+ ALL xenograft cells. More potently, a novel small molecule inhibitor of INPP5D (SHIP1) selectively killed Ph+ ALL xenograft cells through inducing hyper pre-BCR signaling, regardless of TKI resistance. We demonstrate that inhibitory phosphatase signaling represents a potential novel class of therapeutic targets for Ph+ALL. Conclusions: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified ITIM-receptors and phosphatases as members of a potential novel class of therapeutic targets. The concept of pharmacological perturbance of oncogenic signaling equilibrium in leukemia cells by inhibition (e.g. TKI-treatment) or exaggeration of signaling strength (e.g. blockade of ITIM-receptors) may lead to the discovery of multiple additional therapeutic targets and broaden our repertoire of currently available therapeutic intervention. Disclosures No relevant conflicts of interest to declare.

scholarly journals Erk Negative Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

Cancer Cell ◽  
2015 ◽  
Vol 28 (1) ◽  
pp. 114-128 ◽  
Author(s):  
Seyedmehdi Shojaee ◽  
Rebecca Caeser ◽  
Maike Buchner ◽  
Eugene Park ◽  
Srividya Swaminathan ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Feedback Control ◽  
B Cell ◽  
Negative Feedback ◽  
Therapeutic Target ◽  
Cell Transformation ◽  
Lymphoblastic Leukemia ◽  
Negative Feedback Control

CD25 (IL2RA) Orchestrates Negative Feedback Control and Stabilizes Oncogenic Signaling Strength in Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1434-1434 ◽  
Author(s):  
Jae-Woong Lee ◽  
Zhengshan Chen ◽  
Huimin Geng ◽  
Gang Xiao ◽  
Eugene PARK ◽  
...  
Keyword(s):  
Drug Resistance ◽  
B Cells ◽  
Feedback Control ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Negative Feedback ◽  
Cytoplasmic Tail ◽  
Cd25 Expression ◽  
Bcr Signaling ◽  
Negative Feedback Control

Abstract Background and hypothesis: CD25 (IL2RA) represents the α chain of the interleukin 2 receptor on T cells and plays an important role in the maintenance of regulatory T (Treg) cells, hence preventing T cell autoimmunity. In a comprehensive gene expression analysis, we found that CD25 is specifically upregulated by pre-B cell receptor (pre-BCR) signaling during early B cell development and oncogenic tyrosine kinase that mimic pre-BCR signaling (e.g. in Ph+ ALL and Ph-like ALL). In adults with Ph+ ALL (ECOG; MDACC) and children with Ph-like ALL (P9906) patients with CD25 expression at the time of diagnosis have a particularly poor outcome (n=416; P=0.005). For these reasons, we studied the function of CD25 in B cell development and leukemia in a series of genetic experiments. Results: Unlike T cells, CD25 (IL2RA) does not function as IL2 receptor chain in B cells and B-lineage ALL: CD25 expressed on B-lineage cells did not pair with IL2Rb and g-chains and was not responsive to IL2. Il2ra-/- B cells were arrested at the pre-B cell stage with hyperactive pre-BCR downstream signaling including SRC, BTK and ERK. In the presence of CD25, Il2ra+/+ B cells responded to engagement of the pre-BCR with phosphorylation of pre-BCR downstream tyrosine kinases and coordinated release of Ca2+ from cytoplasmic stores. In the absence of CD25 (Il2ra-/-), the pre-BCR signals autonomously, resulting in uncoordinated Ca2+ oscillations of variable duration. While CD25 does not function as IL2 receptor chain in B cells, it coordinates pre-BCR-dependent signal transduction and regulates its intensity. The pre-BCR related tyrosine kinase BTK is phosphorylated by BCR-ABL1 in Ph+ ALL and other tyrosine kinase oncogenes in Ph-like ALL (Chen et al., 2015). Interestingly, overexpression of a constitutively active form of BTK resulted in strong upregulation of CD25 surface expression. Conversely, the BTK-inhibitor ibrutinib abolished CD25 expression suggesting that feedback control between pre-BCR signaling and CD25 requires BTK. The ability of CD25 to stabilize oncogenic signaling strength in Ph+ ALL and Ph-like ALL was important for leukemia-initiation and development of fatal disease. In the absence of CD25, Il2ra-/- ALL cells showed impaired proliferation and colony formation. Serial transplantation experiments revealed a profound defect of Il2ra-/- ALL cells to initiate leukemia. 100-times more cells were required to cause fatal disease. In addition, CD25 expression mediated drug-resistance in ALL cells: In patient-derived pre-B ALL cells with heterogeneous CD25 expression, vincristine selectively induced apoptosis in CD25Low cells but spared CD25High ALL cells. Combination with an anti-CD25 immunotoxin efficiently eradiated CD25High leukemia cells and sensitized the ALL cell population to treatment with vincristine. To elucidate the mechanism of how CD25 coordinates negative feedback control of pre-BCR signaling or its oncogenic mimics, we focused on its short (13aa) cytoplasmic tail, which includes two phosphorylation sites (S268 and T271) that are known substrates for serine/threonine protein kinase, PKCα. To identify cytoplasmic interaction partners of CD25, we overexpressed a Flag-tagged truncated form of CD25 including a myristoylation signal for constitutive membrane localization, transmembrane domain and cytoplasmic tail. Immunoprecipitation (IP; Flag) followed by 2D mass spectrometry revealed strong interactions of PP2A with cytoplasmic tail of CD25. Western blots showed additional strong interactions of the cytoplasmic tail of CD25 with inhibitory phosphatases PTEN, SHP1 and SHIP1. Importantly, reconstitution of myristoylated CD25 tail but not a mutant construct lacking the serine/threonine motif (S268A/T271A) rescued proliferation and survival defects of Il2ra-/- ALL cells. Conclusion: We identified CD25 as a surface receptor that mediates membrane recruitment of PP2A, PTEN, SHP1 and SHIP1, which balances fluctuations in signaling output from a pre-B cell receptor or its oncogenic mimic in ALL cells (e.g. BCR-ABL1 in Ph+ ALL). We propose that CD25-mediated negative feedback control stabilizes oncogenic tyrosine kinase signaling and mediates drug-resistance in Ph+ ALL and Ph-like ALL cells. Targeted inhibition using CD25-directed immunotoxins may be useful in new approaches to overcome drug-resistance in Ph+ ALL and Ph-like ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Gene autoregulation by 3’ UTR-derived bacterial small RNAs

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Mona Hoyos ◽  
Michaela Huber ◽  
Konrad U Förstner ◽  
Kai Papenfort
Keyword(s):  
Feedback Control ◽  
Negative Feedback ◽  
Feedback Regulation ◽  
Transcriptional Level ◽  
Cleavage Sites ◽  
Rnase E ◽  
Rna Seq ◽  
Negative Feedback Regulation ◽  
Small Regulatory Rna ◽  
Negative Feedback Control

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in bacteria relying on small regulatory RNA (sRNA) and the major endoribonuclease, RNase E. TIER-seq analysis (transiently-inactivating-an-endoribonuclease-followed-by-RNA-seq) revealed ~25,000 RNase E-dependent cleavage sites in Vibrio cholerae, several of which resulted in the accumulation of stable sRNAs. Focusing on two examples, OppZ and CarZ, we discovered that these sRNAs are processed from the 3’ untranslated region (3’ UTR) of the oppABCDF and carAB operons, respectively, and base-pair with their own transcripts to inhibit translation. For OppZ, this process also triggers Rho-dependent transcription termination. Our data show that sRNAs from 3’ UTRs serve as autoregulatory elements allowing negative feedback control at the post-transcriptional level.

scholarly journals 702 Dual blockade of the PD-1 checkpoint pathway and the adenosinergic negative feedback signaling pathway with a PD-1/CD73 bispecific antibody for cancer immune therapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A744-A744
Author(s):  
Tingting Zhong ◽  
Zhaoliang Huang ◽  
Xinghua Pang ◽  
Na Chen ◽  
Xiaoping Jin ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Negative Feedback ◽  
Immune Suppression ◽  
Specific Antibody ◽  
Cell Activation ◽  
Immune Therapy ◽  
B Cell Activation

BackgroundCD73 (ecto-5’-nucleotidase) is an ecto-nucleotidase that dephosphorylate AMP to form adenosine. Activation of adenosine signaling pathway in immune cells leads to the suppression of effector functions, down-regulate macrophage phagocytosis, inhibit pro-inflammatory cytokine release, as well as yield aberrantly differentiated dendritic cells producing pro-tumorigenic molecules.1 In the tumor microenvironment, adenosinergic negative feedback signaling facilitated immune suppression is considered an important mechanism for immune evasion of cancer cells.2 3 Combination of CD73 and anti-PD-1 antibody has shown promising activity in suppressing tumor growth. Hence, we developed AK119, an anti- human CD73 monoclonal antibody, and AK123,a bi-specific antibody targeting both PD-1 and CD73 for immune therapy of cancer.MethodsAK119 is a humanized antibody against CD73 and AK123 is a tetrameric bi-specific antibody targeting PD-1 and CD73. Binding assays of AK119 and AK123 to antigens, and antigen expressing cells were performed by using ELISA, Fortebio, and FACS assays. In-vitro assays to investigate the activity of AK119 and AK123 to inhibit CD73 enzymatic activity in modified CellTiter-Glo assay, to induce endocytosis of CD73, and to activate B cells were performed. Assay to evaluate AK123 activity on T cell activation were additionally performed. Moreover, the activities of AK119 and AK123 to mediate ADCC, CDC in CD73 expressing cells were also evaluated.ResultsAK119 and AK123 could bind to its respective soluble or membrane antigens expressing on PBMCs, MDA-MB-231, and U87-MG cells with high affinity. Results from cell-based assays indicated that AK119 and AK123 effectively inhibited nucleotidase enzyme activity of CD73, mediated endocytosis of CD73, and induced B cell activation by upregulating CD69 and CD83 expression on B cells, and showed more robust CD73 blocking and B cell activation activities compared to leading clinical candidate targeting CD73. AK123 could also block PD-1/PD-L1 interaction and enhance T cell activation.ConclusionsIn summary, AK119 and AK123 represent good preclinical biological properties, which support its further development as an anti-cancer immunotherapy or treating other diseases.ReferencesDeaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007; 204:1257–65.Huang S, Apasov S, Koshiba M, Sitkovsky M. Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood. 1997; 90:1600–10.Novitskiy SV, Ryzhov S, Zaynagetdinov R, Goldstein AE, Huang Y, Tikhomirov OY, Blackburn MR, Biaggioni I,Carbone DP, Feoktistov I, et al. Adenosine receptors in regulation of dendritic cell differentiation and function. Blood 2008; 112:1822–31.

scholarly journals The Structure of the PanD/PanZ Protein Complex Reveals Negative Feedback Regulation of Pantothenate Biosynthesis by Coenzyme A

2015 ◽  
Vol 22 (4) ◽  
pp. 492-503 ◽  
Author(s):  
Diana C.F. Monteiro ◽  
Vijay Patel ◽  
Christopher P. Bartlett ◽  
Shingo Nozaki ◽  
Thomas D. Grant ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Protein Complex ◽  
Coenzyme A ◽  
Feedback Regulation ◽  
Negative Feedback Regulation ◽  
Pantothenate Biosynthesis
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