James D. Phelan

Gfi1-cells and circuits: unraveling transcriptional networks of development and disease

Purpose of reviewThe review will integrate current knowledge of transcriptional circuits whose dysregulation leads to autoimmunity, neutropenia and leukemia. Recent findingsGrowth factor independent-1 (Gfi1) is a transcriptional repressor with essential roles in controlling hematopoietic stem cell biology, myeloid and lymphoid differentiation and lymphocyte effector functions. Recent work has suggested that Gfi1 competes or collaborates with other transcription factors to modulate transcription programs and lineage decisions. SummaryGfi1 is central to several transcriptional circuits whose dysregulation leads to abnormal or malignant hematopoiesis. These functional relationships are conserved from Drosophila development. Such conserved pathways represent central oncogenic or ‘gatekeeper’ pathways that are pivotal to understanding the process of cellular transformation, and illustrate key targets for clinical intervention.

Growth Factor Independence 1 Antagonizes a p53-Induced DNA Damage Response Pathway in Lymphoblastic Leukemia

Most patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Because oncogenic signaling activates a p53-dependent DNA damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function because Gfi1 ablation exacerbates p53 responses and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of proapoptotic p53 targets such as Bax, Noxa (Pmaip1), and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias.

Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

ATF3 is a novel regulator of mouse neutrophil migration

Expression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3(-/-) neutrophil recruitment to wild-type lungs. In vitro, ATF3(-/-) neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3(-/-) neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3(-/-) and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis.

Growth factor independent-1 maintains Notch1-dependent transcriptional programming of lymphoid precursors.

Growth factor independent 1 (Gfi1) is a transcriptional repressor originally identified as a gene activated in T-cell leukemias induced by Moloney-murine-leukemia virus infection. Notch1 is a transmembrane receptor that is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL). Gfi1 is an important factor in the initiation and maintenance of lymphoid leukemias and its deficiency significantly impedes Notch dependent initiation of T-ALL in animal models. Here, we show that immature hematopoietic cells require Gfi1 to competently integrate Notch-activated signaling. Notch1 activation coupled with Gfi1 deficiency early in T-lineage specification leads to a dramatic loss of T-cells, whereas activation in later stages leaves development unaffected. In Gfi1 deficient multipotent precursors, Notch activation induces lethality and is cell autonomous. Further, without Gfi1, multipotent progenitors do not maintain Notch1-activated global expression profiles typical for T-lineage precursors. In agreement with this, we find that both lymphoid-primed multipotent progenitors (LMPP) and early T lineage progenitors (ETP) do not properly form or function in Gfi1−/− mice. These defects correlate with an inability of Gfi1−/− progenitors to activate lymphoid genes, including IL7R, Rag1, Flt3 and Notch1. Our data indicate that Gfi1 is required for hematopoietic precursors to withstand Notch1 activation and to maintain Notch1 dependent transcriptional programming to determine early T-lymphoid lineage identity.

Distinct roles of Cdc42 in thymopoiesis and effector and memory T cell differentiation.

Cdc42 of the Rho GTPase family has been implicated in cell actin organization, proliferation, survival, and migration but its physiological role is likely cell-type specific. By a T cell-specific deletion of Cdc42 in mouse, we have recently shown that Cdc42 maintains naïve T cell homeostasis through promoting cell survival and suppressing T cell activation. Here we have further investigated the involvement of Cdc42 in multiple stages of T cell differentiation. We found that in Cdc42−/− thymus, positive selection of CD4+CD8+ double-positive thymocytes was defective, CD4+ and CD8+ single-positive thymocytes were impaired in migration and showed an increase in cell apoptosis triggered by anti-CD3/-CD28 antibodies, and thymocytes were hyporesponsive to anti-CD3/-CD28-induced cell proliferation and hyperresponsive to anti-CD3/-CD28-stimulated MAP kinase activation. At the periphery, Cdc42-deficient naive T cells displayed an impaired actin polymerization and TCR clustering during the formation of mature immunological synapse, and showed an enhanced differentiation to Th1 and CD8+ effector and memory cells in vitro and in vivo. Finally, Cdc42−/− mice exhibited exacerbated liver damage in an induced autoimmune disease model. Collectively, these data establish that Cdc42 is critically involved in thymopoiesis and plays a restrictive role in effector and memory T cell differentiation and autoimmunity.

Abstract PR13: CRISPRi screening with targeted therapeutics classifies functional long non-coding RNAs in DLBCL

While tens of thousands of lncRNAs have been detected in the human genome, the functional significance of the majority of these genes remain untested. Here, we use CRISPR interference (CRISPRi) loss-of-function screening combined with targeted drug treatment to identify oncogenic and tumor suppressive lncRNAs. Integrative analysis of 417 primary human tumor and cell line RNA-seq datasets from Diffuse Large B Cell Lymphoma (DLBCL) identified 1,276 intergenic lncRNA candidates, including 59 de novo assembled transcripts. CRISPRi screening in a genetically diverse panel of 4 DLBCL cell lines identified known ABC DLBCL protein-coding oncogenes (BTK, CD79A) and demonstrated oncogenic function of 247 lncRNAs including MIR155HG and MALAT1. To assign functionally-related pathways to unknown lncRNAs, we performed this CRISPRi screen in the context of drugs with known targets: Ibrutinib (BTK), JQ1 (BRD4), and Etoposide (TOP2A). Significant synergy or resistance to drug treatment was observed with 54 lncRNAs. This ongoing work demonstrates a high-throughput pipeline for functional classification of lncRNAs using synthetic lethality screening in cancer. Citation Format: Dan E. Webster, James D. Phelan, Monica Kasbekar, Arthur L. Shaffer, III, Louis M. Staudt. CRISPRi screening with targeted therapeutics classifies functional long non-coding RNAs in DLBCL [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR13.

Abstract 979: microRNA-mediated leukemia-initiating cell activity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The HoxA9 transcription activator and the Gfi1 transcriptional repressor compete for the regulation of common target genes. We exploited HoxA9 versus Gfi1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of Hox-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b significantly inhibits in vitro colony forming activity, and quantitatively depletes in vivo leukemia-initiating-cell activity of Hox-based leukemias leading to leukemia-free survival of murine AML and significant delay in disease onset in xenograft models. A novel microRNA target identification platform both validates therapeutic intervention and reveals biological impact of disrupting microRNA function. These data establish microRNA as functional effectors of endogenous HoxA9 and Hox-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML. Citation Format: Chinavenmeni S. Velu, Aditya Chaubey, James D. Phelan, Sara Meyer, Shane R. Horman, Mark Wunderlich, Monica L. Guzman, Anil G. Jegga, Nancy J. Zeleznik-Le, Jianjun Chen, James C. Mulloy, Jose A. Cancelas, Craig T. Jordan, Bruce J. Aronow, Guido Marcucci, Balkrishen Bhat, Brian Gebelein, H. Leighton Grimes. microRNA-mediated leukemia-initiating cell activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 979. doi:10.1158/1538-7445.AM2014-979