Acute lymphoid leukemia etiopathogenesis.

Acute lymphoid leukemia (ALL) is a type of hematological neoplasm that affects the precursor cells of strains B, T  and NK, with a higher incidence in the pediatric range. The pathophysiology of ALL is characterized by chromosomal abnormalities and genetic alterations involved in the differentiation and proliferation of lymphoid precursor cells. Despite the lack of information in the literature, it is believed that leukemogenesis originates from a complex interaction between environmental and genetic factors, which combined lead to cellular modifications. Environmental factors have been evaluated as possible predisposing factors in the development of ALL but there are still conflicting results in the world literature. In this context, the aim of the present review is to discuss the major exogenous factors regarding ALL.

IFNγ and iNOS-Mediated Alterations in the Bone Marrow and Thymus and Its Impact on Mycobacterium avium-Induced Thymic Atrophy.

Disseminated infection with the high virulence strain of Mycobacterium avium 25291 leads to progressive thymic atrophy. We previously showed that M. avium-induced thymic atrophy results from increased glucocorticoid levels that synergize with nitric oxide (NO) produced by interferon gamma (IFNγ) activated macrophages. Where and how these mediators act is not understood. We hypothesized that IFNγ and NO promote thymic atrophy through their effects on bone marrow (BM) T cell precursors and T cell differentiation in the thymus. We show that M. avium infection cause a reduction in the percentage and number of common lymphoid progenitors (CLP). Additionally, BM precursors from infected mice show an overall impaired ability to reconstitute thymi of RAGKO mice, in part due to IFNγ. Thymi from infected mice present an IFNγ and NO-driven inflammation. When transplanted under the kidney capsule of uninfected mice, thymi from infected mice are unable to sustain T cell differentiation. Finally, we observed increased thymocyte death via apoptosis after infection, independent of both IFNγ and iNOS; and a decrease on active caspase-3 positive thymocytes, which is not observed in the absence of iNOS expression. Together our data suggests that M. avium-induced thymic atrophy results from a combination of defects mediated by IFNγ and NO, including alterations in the BM T cell precursors, the thymic structure and the thymocyte differentiation.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

Evidence for BCR/ABL1-positive T-cell acute lymphoblastic leukemia arising in an early lymphoid progenitor cell.

BCR-ABL1-positive leukemias have historically been classified as either chronic myelogenous leukemia or Ph+ acute lymphoblastic leukemia. Recent analyses suggest there may be a wider range of subtypes. We report a patient with BCR-ABL1 fusion positive T-cell ALL with a previously undescribed cell distribution of the fusion gene. The examination of sorted cells by fluorescence in situ hybridization showed the BCR-ABL1 fusion in the malignant T cells and a subpopulation of the nonmalignant B cells, but not nonmalignant T cells or myeloid or CD34+ progenitor cells providing evidence that the fusion may have occurred in an early lymphoid progenitor.

The Concerted Action of E2-2 and HEB Is Critical for Early Lymphoid Specification.

The apparition of adaptive immunity in Gnathostomata correlates with the expansion of the E-protein family to encompass E2-2, HEB, and E2A. Within the family, E2-2 and HEB are more closely evolutionarily related but their concerted action in hematopoiesis remains to be explored. Here we show that the combined disruption of E2-2 and HEB results in failure to express the early lymphoid program in Common lymphoid precursors (CLPs) and a near complete block in B-cell development. In the thymus, Early T-cell progenitors (ETPs) were reduced and T-cell development perturbed, resulting in reduced CD4 T- and increased γδ T-cell numbers. In contrast, hematopoietic stem cells (HSCs), erythro-myeloid progenitors, and innate immune cells were unaffected showing that E2-2 and HEB are dispensable for the ancestral hematopoietic lineages. Taken together, this E-protein dependence suggests that the appearance of the full Gnathostomata E-protein repertoire was critical to reinforce the gene regulatory circuits that drove the emergence and expansion of the lineages constituting humoral immunity.

An ST2-dependent role of bone marrow-derived group 2 innate lymphoid cells in pulmonary fibrosis.

Recent evidence supports that bone marrow (BM)-derived hematopoietic progenitor cells play an important role in lung injury and fibrosis. While these cells give rise to multiple cell types, the ST2 (Il1rl1)-expressing group 2 innate lymphoid cells (ILC2s) derived from BM progenitors have been implicated in tissue repair and remodeling, including in lung fibrosis. To further investigate the precise role of BM-derived ILC2s in the pathogenesis of fibrotic lung disease, their importance in the bleomycin-induced lung fibrosis model was evaluated by analyzing the effects of selective ST2 deficiency in the BM compartment. The results showed that while ST2-sufficient control mice exhibited activation of lung IL-33/ST2 signaling, ILC2 recruitment, IL-13 induction, and fibrosis, these responses were significantly diminished in ST2-deficient-BM chimera mice, with selective loss of ST2 expression only in the BM. This diminished response to bleomycin was similar to that seen in ST2 global knockout mice, suggesting the predominant importance of ST2 from the BM compartment. In wild-type mice, ILC2 recruitment to the lung was accompanied by a concomitant decrease in ST2+ BM cells. ST2-deficient BM cells were unresponsive to IL-33-induced ILC2 maturation. Finally, lineage-negative wild-type, but not ST2-deficient BM cells from bleomycin-treated mice stimulated lung fibroblast type I collagen expression, which was associated with elevated TGFß expression in the BM cells. Taken together, these findings suggested that the BM-derived ILC2s were recruited to fibrotic lung through the IL-33/ST2 pathway, and contributed to fibroblast activation to promote lung fibrosis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Direct role of FLT3 in regulation of early lymphoid progenitors.

Given that FLT3 expression is highly restricted on lymphoid progenitors, it is possible that the established role of FLT3 in the regulation of B and T lymphopoiesis reflects its high expression and role in regulation of lymphoid-primed multipotent progenitors (LMPPs) or common lymphoid progenitors (CLPs). We generated a Flt3 conditional knock-out (Flt3fl/fl) mouse model to address the direct role of FLT3 in regulation of lymphoid-restricted progenitors, subsequent to turning on Rag1 expression, as well as potentially ontogeny-specific roles in B and T lymphopoiesis. Our studies establish a prominent and direct role of FLT3, independently of the established role of FLT3 in regulation of LMPPs and CLPs, in regulation of fetal as well as adult early B cell progenitors, and the early thymic progenitors (ETPs) in adult mice but not in the fetus. Our findings highlight the potential benefit of targeting poor prognosis acute B-cell progenitor leukaemia and ETP leukaemia with recurrent FLT3 mutations using clinical FLT3 inhibitors.

Mechanisms underlying the heterogeneity of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders in which recurrent chromosome abnormalities and gene mutations define a clonal hematopoiesis. The MDS-initiating cell is a rare HSC which transmits the genetic abnormalities to its myeloid and lymphoid progeny. The heterogeneity of MDS phenotypes could be linked to the diversity of genetic events involving epigenetic regulators, chromatin modifiers, splicing factors, transcription factors and signaling adaptors, the various combinations and order of mutations in cooperating genes, and the variegation of clonal hematopoietic hierarchy. Usually, epigenetic and splicing gene mutations occur first. A combination of one epigenetic event with a splicing gene alteration is frequent. The HSC compartment is invaded by a dominant and few minor clones organized linearly or with a branched architecture. The dominant clone containing the first initiating mutations produces myeloid and lymphoid lineages in transplanted immune-deficient mice. The mutations confer a selective advantage to myeloid progenitors at the expense of lymphoid progenitors. In the context of differentiation, one mutation may favor the amplification of granulo-monocytic progenitor, which drives the transformation into acute myeloid leukemia. Understanding the hierarchy of mutations provides insights on the mechanism of transformation. Investigation of mutation pattern and distribution along the hematopoietic tree may influence the therapeutic decision for targeted therapy.

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

Acute myeloid leukemia derived from lympho-myeloid clonal hematopoiesis.

We studied acute myeloid leukemia (AML) patients with lympho-myeloid clonal hematopoiesis (LM-CH), defined by the presence of DNA methyltransferase 3A (DNMT3A) mutations in both the myeloid and lymphoid T-cell compartment. Diagnostic, complete remission (CR) and relapse samples were sequenced for 34 leukemia-related genes in 171 DNMT3A mutated adult AML patients. AML with LM-CH was found in 40 patients (23%) and was associated with clonal hematopoiesis of indeterminate potential years before AML, older age, secondary AML and more frequent MDS-type co-mutations (TET2, RUNX1 and EZH2). In 82% of AML patients with LM-CH, the preleukemic clone was refractory to chemotherapy and was the founding clone for relapse. Both LM-CH and non-LM-CH MRD-positive AML patients who achieved CR had a high risk of relapse after 10 years (75% and 75%, respectively) compared with patients without clonal hematopoiesis in CR with negative MRD (27% relapse rate). Long-term survival of patients with LM-CH was only seen after allogeneic hematopoietic stem cell transplantation (HSCT). We define AML patients with LM-CH as a distinct high-risk group of AML patients that can be identified at diagnosis through mutation analysis in T cells and should be considered for HSCT.

Loss of UHRF2 expression is associated with human neoplasia, promoter hypermethylation, decreased 5-hydroxymethylcytosine, and high proliferative activity.

Ubiquitin-like with PHD and ring finger domains 2 (UHRF2) binds to 5-hydroxymethylcytosine (5hmC), a DNA base involved in tissue development, but it is unknown how their distribution compares with each other in normal and malignant human tissues. We used IHC on human tumor specimens (160 from 19 tumor types) or normal tissue to determine the expression and distribution of UHRF2, Ki-67, and 5hmC. We also examined UHRF2 expression in cord blood progenitors and compared its expression to methylation status in 6 leukemia cell lines and 15 primary human leukemias. UHRF2 is highly expressed, paralleling that of 5hmC, in most non-neoplastic, differentiated tissue with low Ki-67 defined proliferative activity. UHRF2 is expressed in common lymphoid progenitors and mature lymphocytes but not common myeloid progenitors or monocytes. In contrast, UHRF2 immunostaining in human cancer tissues revealed widespread reduction or abnormal cytoplasmic localization which correlated with a higher Ki-67 and reduced 5hmC. UHRF2 expression is reduced in some leukemia cell lines, this correlates with promoter hypermethylation, and similar UHRF2 methylation profiles are seen in primary human leukemia samples. Thus, UHRF2 and 5hmC are widely present in differentiated human tissues, and UHRF2 protein is poorly expressed or mislocalized in diverse human cancers.

Generation and characterization of RAG2 knockout pigs as animal model for severe combined immunodeficiency.

Pigs with severe combined immunodeficiency (SCID) are versatile animal models for human medical research because of their biological similarities to humans, suitable body size, and longevity for practical research. SCID pigs with defined mutation(s) can be an invaluable tool for research on porcine immunity. In this study, we produced RAG2-knockout pigs via somatic cell nuclear transfer and analyzed their phenotype. The V(D)J recombination processes were confirmed as being inactivated. They consistently lacked mature T and B cells but had substantial numbers of cells considered to be T- or B-cell progenitors as well as NK cells. They also lacked thymic medulla and lymphoid aggregations in the spleen, mesenteric lymph nodes, and ileal Peyer's patches. We showed more severe immunological defects in the RAG2 and IL2RG double-knockout pig through this study. Thus, SCID pigs could be promising animal models not only for translational medical research but also for immunological studies of pigs themselves.

Increased uNK Progenitor Cells in Women With Endometriosis and Infertility are Associated With Low Levels of Endometrial Stem Cell Factor.

PROBLEM: Uterine natural killer (uNK) cells play a significant role in successful human pregnancy. Having previously demonstrated uNK cell progenitors in human endometrium, we hypothesized that abnormal uNK cell maturation contributes to infertility in women with endometriosis. We aimed to characterize uNK cells at different developmental stages in women with and without endometriosis and to investigate possible mechanisms to explain any differences. METHOD OF STUDY: We characterized uNK cell development in women with and without endometriosis using flow cytometry, protein array and in vitro experiments. RESULTS: We found increased proportions of uNK cells at developmental stages 1 and 2 in endometrium from women with endometriosis (n = 36; mean = 21.2%) when compared with healthy fertile women (n = 9; mean = 7.0%). Protein array analysis revealed significantly lower levels of stem cell factor (SCF) in the eutopic endometrium of women with endometriosis when compared to healthy women. Addition of SCF to endometrial progenitor cells in vitro restored uNK cell maturation. CONCLUSION: We have shown that women with endometriosis have low levels of endometrial SCF, which we hypothesize contributes to abnormal maturation of local uNK cell populations. This defect may also compromise embryo implantation and hence contribute to endometriosis-associated infertility. SCF replacement may be a new therapeutic approach.

CD93 Marks a Non-Quiescent Human Leukemia Stem Cell Population and Is Required for Development of MLL-Rearranged Acute Myeloid Leukemia.

Leukemia stem cells (LSCs) are thought to share several properties with hematopoietic stem cells (HSCs), including cell-cycle quiescence and a capacity for self-renewal. These features are hypothesized to underlie leukemic initiation, progression, and relapse, and they also complicate efforts to eradicate leukemia through therapeutic targeting of LSCs without adverse effects on HSCs. Here, we show that acute myeloid leukemias (AMLs) with genomic rearrangements of the MLL gene contain a non-quiescent LSC population. Although human CD34(+)CD38(-) LSCs are generally highly quiescent, the C-type lectin CD93 is expressed on a subset of actively cycling, non-quiescent AML cells enriched for LSC activity. CD93 expression is functionally required for engraftment of primary human AML LSCs and leukemogenesis, and it regulates LSC self-renewal predominantly by silencing CDKN2B, a major tumor suppressor in AML. Thus, CD93 expression identifies a predominantly cycling, non-quiescent leukemia-initiating cell population in MLL-rearranged AML, providing opportunities for selective targeting and eradication of LSCs.

The autotaxin-LPA2 GPCR axis is modulated by γ-irradiation and facilitates DNA damage repair.

In this study we characterized the effects of radiation injury on the expression and function of the autotaxin (ATX)-LPA2 GPCR axis. In IEC-6 crypt cells and jejunum enteroids quantitative RT-PCR showed a time- and dose-dependent upregulation of lpa2 in response to γ-irradiation that was abolished by mutation of the NF-κB site in the lpa2 promoter or by inhibition of ATM/ATR kinases with CGK-733, suggesting that lpa2 is a DNA damage response gene upregulated by ATM via NF-κB. The resolution kinetics of the DNA damage marker γ-H2AX in LPA-treated IEC-6 cells exposed to γ-irradiation was accelerated compared to vehicle, whereas pharmacological inhibition of LPA2 delayed the resolution of γ-H2AX. In LPA2-reconstituted MEF cells lacking LPA1&3 the levels of γ-H2AX decreased rapidly, whereas in Vector MEF were high and remained sustained. Inhibition of ERK1&2 or PI3K/AKT signaling axis by pertussis toxin or the C311A/C314A/L351A mutation in the C-terminus of LPA2 abrogated the effect of LPA on DNA repair. LPA2 transcripts in Lin(-)Sca-1(+)c-Kit(+) enriched for bone marrow stem cells were 27- and 5-fold higher than in common myeloid or lymphoid progenitors, respectively. Furthermore, after irradiation higher residual γ-H2AX levels were detected in the bone marrow or jejunum of irradiated LPA2-KO mice compared to WT mice. We found that γ-irradiation increases plasma ATX activity and LPA level that is in part due to the previously established radiation-induced upregulation of TNFα. These findings identify ATX and LPA2 as radiation-regulated genes that appear to play a physiological role in DNA repair.

The Yin and Yang aspects of IL-27 in induction of cancer-specific T-cell responses and immunotherapy.

Accumulating evidences from animal studies have indicated that both endogenous and exogenous IL-27, an IL-12 family of cytokine, can increase antitumor T-cell activities and inhibit tumor growth. IL-27 can modulate Treg responses, and program effector T cells into a unique T-effector stem cell (TSEC) phenotype, which enhances T-cell survival in the tumor microenvironment. However, animal studies also suggest that IL-27 induces molecular pathways such as IL-10, PD-L1 and CD39, which may downregulate tumor-specific T-cell responses. In this review paper, we will discuss the Yin and Yang aspects of IL-27 in the induction of tumor-specific T-cell responses, and the potential impacts of these functions of IL-27 in the design of cancer immunotherapy.

[About possible additions to actual scheme of normal blood formation on the basis of study of leukemic blast cells].

The issue of introduction of number of additions into actual scheme of blood formation is discussed. The long standing experience of laboratory diagnostic of oncologic hematological diseases in adults and children and the analysis of published data about normal blood formation are involved into consideration. The existence is surmised of common oligo-linear precursors for B-lymphocytes and monocytes, natural killer cells and monocytes and common cell-precursor of T-lymphocytes and dendrite cells as well. At the same time, the issue concerning the existence of human common cell-precursor of lymphization capable of differentiating into Band T-lymphocytes and natural killer cells is disputable.