Unveiling the complexity of transcription factor networks in hematopoietic stem cells: implications for cell therapy and hematological malignancies

The functionality and longevity of hematopoietic tissue is ensured by a tightly controlled balance between self-renewal, quiescence, and differentiation of hematopoietic stem cells (HSCs) into the many different blood lineages. Cell fate determination in HSCs is influenced by signals from extrinsic factors (e.g., cytokines, irradiation, reactive oxygen species, O2 concentration) that are translated and integrated by intrinsic factors such as Transcription Factors (TFs) to establish specific gene regulatory programs. TFs also play a central role in the establishment and/or maintenance of hematological malignancies, highlighting the need to understand their functions in multiple contexts. TFs bind to specific DNA sequences and interact with each other to form transcriptional complexes that directly or indirectly control the expression of multiple genes. Over the past decades, significant research efforts have unraveled molecular programs that control HSC function. This, in turn, led to the identification of more than 50 TF proteins that influence HSC fate. However, much remains to be learned about how these proteins interact to form molecular networks in combination with cofactors (e.g. epigenetics factors) and how they control differentiation, expansion, and maintenance of cellular identity. Understanding these processes is critical for future applications particularly in the field of cell therapy, as this would allow for manipulation of cell fate and induction of expansion, differentiation, or reprogramming of HSCs using specific cocktails of TFs. Here, we review recent findings that have unraveled the complexity of molecular networks controlled by TFs in HSCs and point towards possible applications to obtain functional HSCs ex vivo for therapeutic purposes including hematological malignancies. Furthermore, we discuss the challenges and prospects for the derivation and expansion of functional adult HSCs in the near future

Vascular endothelial growth factor directly inhibits primitive neural stem cell survival but promotes definitive neural stem cell survival

There are two types of neural stem cells (NSCs). Primitive NSCs [leukemia inhibitory factor (LIF) dependent but exogenous fibroblast growth factor (FGF) 2 independent] can be derived from mouse embryonic stem (ES) cells in vitro and from embryonic day 5.5 (E5.5) to E7.5 epiblast and E7.5-E8.5 neuroectoderm in vivo. Definitive NSCs (LIF independent but FGF2 dependent) first appear in the E8.5 neural plate and persist throughout life. Primitive NSCs give rise to definitive NSCs. Loss and gain of functions were used to study the role of vascular endothelial growth factor (VEGF)-A and its receptor, Flk1, in NSCs. The numbers of Flk1 knock-out mice embryo-derived and ES cell-derived primitive NSCs were increased because of the enhanced survival of primitive NSCs. In contrast, neural precursor-specific, Flk1 conditional knock-out mice-derived, definitive NSCs numbers were decreased because of the enhanced cell death of definitive NSCs. These effects were not observed in cells lacking Flt1, another VEGF receptor. In addition, the cell death stimulated by VEGF-A of primitive NSC and the cell survival stimulated by VEGF-A of definitive NSC were blocked by Flk1/Fc-soluble receptors and VEGF-A function-blocking antibodies. These VEGF-A phenotypes also were blocked by inhibition of the downstream effector nuclear factor kappa B (NF-kappa B). Thus, both the cell death of primitive NSC and the cell survival of definitive NSC induced by VEGF-A stimulation are mediated by bifunctional NF-kappa B effects. In conclusion, VEGF-A function through Flk1 mediates survival (and not proliferative or fate change) effects on NSCs, specifically

STAT5 activation promotes progression and chemotherapy-resistance in early T-cell precursor acute lymphoblastic leukemia

IL-7 supports the growth and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL), particularly the early T-cell precursor subtype (ETP-ALL), which frequently has activating mutations of IL-7 signaling. STAT5 is an attractive therapeutic target because it is almost universally activated in ETP-ALL, even in the absence of mutations of upstream activators such as the IL-7R, JAK and FLT3. To examine the role of activated STAT5 in ETP-ALL, we have used a Lmo2-transgenic (Lmo2Tg) mouse model in which we can monitor chemoresistant pre-leukemia (pre-LSCs) and leukemia stem cells (LSCs) that drive T-ALL development and relapse following chemotherapy. Using IL-7R-deficient Lmo2Tg mice, we show that IL-7 signaling was not required for the formation of pre-LSCs but essential for their expansion and clonal evolution into LSCs to generate T-ALL. Activated STAT5B was sufficient for the development of T-ALL in IL-7R; Lmo2Tg mice, indicating that inhibition of STAT5 is required to block the supportive signals provided by IL-7. To further understand the role of activated STAT5 in LSCs of ETP-ALL, we developed a new transgenic mouse that enables T-cell specific and doxycycline-inducible expression of the constitutively activated STAT5B1∗6 mutant. Expression of STAT5B1∗6 in T-cells had no effect alone but promoted expansion and chemoresistance of LSCs in Lmo2Tg mice. Pharmacologic inhibition of STAT5 with Pimozide induced differentiation and loss of LSCs, whilst enhancing response to chemotherapy. Furthermore, Pimozide significantly reduced leukemia burden in vivo and overcame chemoresistance of patient-derived ETP-ALL xenografts. Overall, our results demonstrate that STAT5 is an attractive therapeutic target for eradicating LSCs in ETP-ALL

ZEB2 and LMO2 drive immature T-cell lymphoblastic leukemia via distinct oncogenic mechanisms

ZEB1 and ZEB2 are structurally related E-box binding homeobox transcription factors that induce epithelial to mesenchymal transitions during development and disease. As such, they regulate cancer cell invasion, dissemination and metastasis of solid tumors. In addition, their expression is associated with the gain of cancer stem cell properties and resistance to therapy. Using conditional loss-of-function mice, we previously demonstrated that Zeb2 also plays pivotal roles in hematopoiesis, controlling important cell fate decisions, lineage commitment and fidelity. In addition, upon Zeb2 overexpression, mice spontaneously develop immature T-cell lymphoblastic leukemia. Here we show that pre-leukemic Zeb2-overexpressing thymocytes are characterized by a differentiation delay at beta-selection due to aberrant activation of the interleukin-7 receptor signaling pathway. Notably, and in contrast to Lmo2-overexpressing thymocytes, these pre-leukemic Zeb2-overexpressing T-cell progenitors display no acquired self-renewal properties. Finally, Zeb2 activation in more differentiated T-cell precursor cells can also drive malignant T-cell development, suggesting that the early T-cell differentiation delay is not essential for Zeb2-mediated leukemic transformation. Altogether, our data suggest that Zeb2 and Lmo2 drive malignant transformation of immature T-cell progenitors via distinct molecular mechanisms

Expressed repetitive elements are broadly applicable reference targets for normalization of reverse transcription-qPCR data in mice

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data

Transcriptional repressor ZEB2 promotes terminal differentiation of CD8⁺ effector and memory T cell populations during infection

ZEB2 is a multi-zinc-finger transcription factor known to play a significant role in early neurogenesis and in epithelial-mesenchymal transition-dependent tumor metastasis. Although the function of ZEB2 in T lymphocytes is unknown, activity of the closely related family member ZEB1 has been implicated in lymphocyte development. Here, we find that ZEB2 expression is up-regulated by activated T cells, specifically in the KLRG1(hi) effector CD8(+) T cell subset. Loss of ZEB2 expression results in a significant loss of antigen-specific CD8(+) T cells after primary and secondary infection with a severe impairment in the generation of the KLRG1(hi) effector memory cell population. We show that ZEB2, which can bind DNA at tandem, consensus E-box sites, regulates gene expression of several E-protein targets and may directly repress Il7r and Il2 in CD8(+) T cells responding to infection. Furthermore, we find that T-bet binds to highly conserved T-box sites in the Zeb2 gene and that T-bet and ZEB2 regulate similar gene expression programs in effector T cells, suggesting that T-bet acts upstream and through regulation of ZEB2. Collectively, we place ZEB2 in a larger transcriptional network that is responsible for the balance between terminal differentiation and formation of memory CD8(+) T cells

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner

Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection

Natural killer (NK) cell maturation is a tightly controlled process that endows NK cells with functional competence and the capacity to recognize target cells. Here, we found that the transcription factor (TF) Zeb2 was the most highly induced TF during NK cell maturation. Zeb2 is known to control epithelial to mesenchymal transition, but its role in immune cells is mostly undefined. Targeted deletion of Zeb2 resulted in impaired NK cell maturation, survival, and exit from the bone marrow. NK cell function was preserved, but mice lacking Zeb2 in NK cells were more susceptible to B16 melanoma lung metastases. Reciprocally, ectopic expression of Zeb2 resulted in a higher frequency of mature NK cells in all organs. Moreover, the immature phenotype of Zeb2(-/-) NK cells closely resembled that of Tbx21(-/-) NK cells. This was caused by both a dependence of Zeb2 expression on T-bet and a probable cooperation of these factors in gene regulation. Transgenic expression of Zeb2 in Tbx21(-/-) NK cells partially restored a normal maturation, establishing that timely induction of Zeb2 by T-bet is an essential event during NK cell differentiation. Finally, this novel transcriptional cascade could also operate in human as T-bet and Zeb2 are similarly regulated in mouse and human NK cells