Jennifer Fraszczak

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.

Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells

The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin-c-Kit+ fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways.

Threshold Levels of Gfi1 Maintain E2A Activity for B Cell Commitment via Repression of Id1.

A regulatory circuit that controls myeloid versus B lymphoid cell fate in hematopoietic progenitors has been proposed, in which a network of the transcription factors Egr1/2, Nab, Gfi1 and PU.1 forms the core element. Here we show that a direct link between Gfi1, the transcription factor E2A and its inhibitor Id1 is a critical element of this regulatory circuit. Our data suggest that a certain threshold of Gfi1 is required to gauge E2A activity by adjusting levels of Id1 in multipotent progenitors, which are the first bipotential myeloid/lymphoid-restricted progeny of hematopoietic stem cells. If Gfi1 levels are high, Id1 is repressed enabling E2A to activate a specific set of B lineage genes by binding to regulatory elements for example the IL7 receptor gene. If Gfi1 levels fall below a threshold, Id1 expression increases and renders E2A unable to function, which prevents hematopoietic progenitors from engaging along the B lymphoid lineage.

The role of the transcriptional repressor growth factor independent 1 in the formation of myeloid cells

Purpose of review Growth factor independent 1 (Gfi1) is a transcriptional repressor that plays multiple roles during myeloid commitment and development. Gfi1-deficient mice lack granulocytes, accumulate aberrant monocytes and show a hyperactivity of macrophages toward bacterial cell wall components. Since these initial findings, numerous additional studies have confirmed the role of Gfi1 in myeloid differentiation from hematopoietic stem cells and multipotent progenitors to bipotential lymphoid/myeloid precursors and myeloid effector cells. This review will summarize the existing knowledge concerning the mechanisms through which Gfi1 exerts these actions and will highlight recent insights into its additional implication in myeloid malignancies. Recent findings Gfi1 has more recently been implicated in myeloid malignancies, in particular in myelodysplasia, myeloproliferative neoplasms and in acute myeloid leukemia, a fatal disease, which is essentially treated today the same way as 30 years ago. Summary Recent findings on the role of Gfi1 in myeloid malignancies together with the knowledge base built over many years on this molecule may help us to find new ways to predict the progression of acute myeloid leukemia and to design more efficient epigenetic drugs to treat this disease.

GFI1 facilitates efficient DNA repair by regulating PRMT1 dependent methylation of MRE11 and 53BP1

GFI1 is a transcriptional regulator expressed in lymphoid cells, and an “oncorequisite” factor required for development and maintenance of T-lymphoid leukemia. GFI1 deletion causes hypersensitivity to ionizing radiation, for which the molecular mechanism remains unknown. Here, we demonstrate that GFI1 is required in T cells for the regulation of key DNA damage signaling and repair proteins. Specifically, GFI1 interacts with the arginine methyltransferase PRMT1 and its substrates MRE11 and 53BP1. We demonstrate that GFI1 enables PRMT1 to bind and methylate MRE11 and 53BP1, which is necessary for their function in the DNA damage response. Thus, our results provide evidence that GFI1 can adopt non-transcriptional roles, mediating the post-translational modification of proteins involved in DNA repair. These findings have direct implications for treatment responses in tumors overexpressing GFI1 and suggest that GFI1’s activity may be a therapeutic target in these malignancies.The transcription factor GFI1 mediates the DNA damage response (DDR) of T cells through a yet unknown mechanism. Here the authors show that GFI1 can adopt non-transcriptional roles during DDR, enabling PRMT1 to bind and methylate the DNA repair proteins MRE11 and 53BP1.

Reduced expression but not deficiency of GFI1 causes a fatal myeloproliferative disease in mice

Growth factor independent 1 (Gfi1) controls myeloid differentiation by regulating gene expression and limits the activation of p53 by facilitating its de-methylation at Lysine 372. In human myeloid leukemia, low GFI1 levels correlate with an inferior prognosis. Here, we show that knockdown (KD) of Gfi1 in mice causes a fatal myeloproliferative disease (MPN) that could progress to leukemia after additional mutations. Both KO and KD mice accumulate myeloid cells that show signs of metabolic stress and high levels of reactive oxygen species. However, only KO cells have elevated levels of Lysine 372 methylated p53. This suggests that in contrast to absence of GFI1, KD of GFI1 leads to the accumulation of myeloid cells because sufficient amount of GFI1 is present to impede p53-mediated cell death, leading to a fatal MPN. The combination of myeloid accumulation and the ability to counteract p53 activity under metabolic stress could explain the role of reduced GF1 expression in human myeloid leukemia.

Abstract 997: Loss of Miz-1 increases latency of T-ALL by preventing induction of autophagy

The Myc-Interacting Zinc-finger protein-1 (Miz-1) can bind to the proto-oncogene c-Myc and the Miz-1/c-Myc complex can regulate the transcription of specific target genes. Miz-1 contains a BTB/POZ domain and thirteen zinc finger domains that mediate binding to DNA and its interaction with partner proteins. While Miz-1 itself functions as a transcriptional transactivator, it enables c-Myc to act as a transcriptional repressor of genes negatively regulating cell cycle progression. We have previously reported that Miz-1 plays an essential role during early stages of T cell development by regulating the translation of p53 via the ribosomal protein L22. Based on this function of Miz-1 in T cell development, we have decided to investigate a potential role of Miz-1 in the development of T cell leukemia or lymphoma. We have used several different experimental models to induce T-ALL in mice that are conditionally deficient for Miz-1. We used non-acute transforming retroviruses, such as Moloney Murine Leukemia Virus (MMLV), which induces T-cell leukemia by activating known oncogenes, including Gfi1, Pim1 and c-Myc via proviral insertion. We also used ENU mutagenesis, which induces T-cell leukemia through activation of the Ras pathway. We observed that Miz-1 is required for the development of T cell leukemia regardless of whether it is induced by MMLV or ENU, since Miz-1-deficient mice succumb to the disease with a significantly longer latency period than WT animals. Importantly, during tumorigenesis a strong counter selection occurs against the loss of Miz-1. Interestingly, MycV394D transgenic animals, carrying a c-Myc protein that can no longer interact directly with Miz-1, develop tumors at a rate comparable to WT mice, indicating that a function of Miz-1 is required for T cell tumorigenesis that is independent of its interaction with c-Myc. Transcriptome analyses showed that Miz-1 deficiency correlated with a decrease of expression in autophagy-related genes, consistent with earlier reports that Miz-1 is necessary for the induction of this pathway. ChIP-seq confirmed that Miz-1 binds to the promoters of these autophagy-related genes, indicating that these genes are activated in the presence of Miz-1, also consistent with previous reports. Furthermore, Miz-1-deficient tumors undergo higher levels of apoptosis than WT tumors. Based on these results, we conclude that Miz-1 is essential for the efficient development of T-ALL and that loss of Miz-1 significantly hinders T-ALL development in part through the inability of these cells to induce autophagy. Citation Format: Marissa Rashkovan, Charles Vadnais, Julie Ross, Jennifer Fraszczak, Tarik Moroy. Loss of Miz-1 increases latency of T-ALL by preventing induction of autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 997. doi:10.1158/1538-7445.AM2015-997