Potential impact of NOTCH1 activation on venetoclax sensitivity in chronic lymphocytic Leukaemia: In vitro insights and clinical implications.

Despite significant progress in treating chronic lymphocytic leukaemia (CLL), resistance to therapy remains challenging. NOTCH1 activation, common in CLL, confers adverse prognosis. This study explores the impact of NOTCH1 signalling on venetoclax sensitivity in vitro. Although NOTCH1 activation minimally impaired the susceptibility of CLL cells to venetoclax, ex vivo cell competition studies reveal that cells with constitutive NOTCH1 activation outgrew their wild-type counterparts in the presence of ongoing venetoclax exposure. Our findings suggest that while NOTCH1 activation is insufficient to confer venetoclax refractoriness, there is enhanced potential for cells with NOTCH1 activation to escape and thus become fully resistant to venetoclax.

The Haematopoietically-expressed homeobox transcription factor: roles in development, physiology and disease.

The Haematopoietically expressed homeobox transcription factor (Hhex) is a transcriptional repressor that is of fundamental importance across species, as evident by its evolutionary conservation spanning fish, amphibians, birds, mice and humans. Indeed, Hhex maintains its vital functions throughout the lifespan of the organism, beginning in the oocyte, through fundamental stages of embryogenesis in the foregut endoderm. The endodermal development driven by Hhex gives rise to endocrine organs such as the pancreas in a process which is likely linked to its role as a risk factor in diabetes and pancreatic disorders. Hhex is also required for the normal development of the bile duct and liver, the latter also importantly being the initial site of haematopoiesis. These haematopoietic origins are governed by Hhex, leading to its crucial later roles in definitive haematopoietic stem cell (HSC) self-renewal, lymphopoiesis and haematological malignancy. Hhex is also necessary for the developing forebrain and thyroid gland, with this reliance on Hhex evident in its role in endocrine disorders later in life including a potential role in Alzheimer's disease. Thus, the roles of Hhex in embryological development throughout evolution appear to be linked to its later roles in a variety of disease processes.

Emerging role of dual biologic therapy for the treatment of inflammatory bowel disease.

Biologic agents have now been used in the management of inflammatory bowel disease (IBD) for many years where experience, expertise and confidence in their use has developed over time. In the United Kingdom, there are well established guidelines and recommendations for both single agent biologic treatments, and with combination therapy of a biologic agent with a small molecule agent in maintenance therapy. In recent times, there has been increasing interest and experience using dual biologic therapy (DBT) in IBD, primarily in difficult to treat and refractory cases with high disease burden. However, published data on use, experience and safety profiles is limited and large-scale studies remain low in number in this developing area. We therefore aim to present a summary and review of the available published data in this area to help us better understand the emerging role of DBT in IBD.

Overexpression of Lmo2 initiates T-lymphoblastic leukemia via impaired thymocyte competition.

Cell competition has recently emerged as an important tumor suppressor mechanism in the thymus that inhibits autonomous thymic maintenance. Here, we show that the oncogenic transcription factor Lmo2 causes autonomous thymic maintenance in transgenic mice by inhibiting early T cell differentiation. This autonomous thymic maintenance results in the development of self-renewing preleukemic stem cells (pre-LSCs) and subsequent leukemogenesis, both of which are profoundly inhibited by restoration of thymic competition or expression of the antiapoptotic factor BCL2. Genomic analyses revealed the presence of Notch1 mutations in pre-LSCs before subsequent loss of tumor suppressors promotes the transition to overt leukemogenesis. These studies demonstrate a critical role for impaired cell competition in the development of pre-LSCs in a transgenic mouse model of T cell acute lymphoblastic leukemia (T-ALL), implying that this process plays a role in the ontogeny of human T-ALL.

REC drives recombination to repair double-strand breaks in animal mtDNA.

Mechanisms that safeguard mitochondrial DNA (mtDNA) limit the accumulation of mutations linked to mitochondrial and age-related diseases. Yet, pathways that repair double-strand breaks (DSBs) in animal mitochondria are poorly understood. By performing a candidate screen for mtDNA repair proteins, we identify that REC-an MCM helicase that drives meiotic recombination in the nucleus-also localizes to mitochondria in Drosophila. We show that REC repairs mtDNA DSBs by homologous recombination in somatic and germline tissues. Moreover, REC prevents age-associated mtDNA mutations. We further show that MCM8, the human ortholog of REC, also localizes to mitochondria and limits the accumulation of mtDNA mutations. This study provides mechanistic insight into animal mtDNA recombination and demonstrates its importance in safeguarding mtDNA during ageing and evolution.

Numerical Analysis of Microchannels Designed for Heat Sinks.

Conjugate heat transfer is numerically investigated using a three-dimensional computational fluid dynamics approach in various microchannel geometries to identify a high-performance cooling method for piezoelectric ceramic stacks and spindle units in high-precision machines. Straight microchannels with rectangular cross sections are first considered, showing the performance limitations of decreasing the size of the microchannels, so other solutions are needed for high applied heat fluxes. Next, many microchannel designs, focusing on streamwise geometric variation, are compared to straight channels to assess their performances. Sinusoidally varying channels produce the highest heat transfer rates of those studied. Thus, their optimization is considered at a channel width and height of 35 and 100 µm, respectively. Heat transfer increases as the amplitude and spatial frequencies of the channels increase due to increased interfacial surface area and enhanced Dean flow. The highest performance efficiencies are observed at intermediate levels of amplitude and frequency, with efficiency decreasing as these geometric parameters are increased further at the onset of flow separation. The sinusoidal channel geometries are then optimized with respect to minimizing the system's pressure drop for all applied heat fluxes between 5690 and 6510 kW/m2. Doing so created an optimal geometry curve and showed that all geometries in this region had amplitudes close to 40 µm. Therefore, imposing a fixed heat flux requirement for a case study of cooling piezoelectric ceramics, the optimized sinusoidal geometry decreases the system pressure drop by 79% relative to a straight channel while maintaining a larger minimum feature size.

T-ALL can evolve to oncogene independence.

The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor.

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Hhex Directly Represses BIM-Dependent Apoptosis to Promote NK Cell Development and Maintenance.

Hhex encodes a homeobox transcriptional regulator important for embryonic development and hematopoiesis. Hhex is highly expressed in NK cells, and its germline deletion results in significant defects in lymphoid development, including NK cells. To determine if Hhex is intrinsically required throughout NK cell development or for NK cell function, we generate mice that specifically lack Hhex in NK cells. NK cell frequency is dramatically reduced, while NK cell differentiation, IL-15 responsiveness, and function at the cellular level remain largely normal in the absence of Hhex. Increased IL-15 availability fails to fully reverse NK lymphopenia following conditional Hhex deletion, suggesting that Hhex regulates developmental pathways extrinsic to those dependent on IL-15. Gene expression and functional genetic approaches reveal that Hhex regulates NK cell survival by directly binding Bcl2l11 (Bim) and repressing expression of this key apoptotic mediator. These data implicate Hhex as a transcriptional regulator of NK cell homeostasis and immunity.

The EMT modulator SNAI1 contributes to AML pathogenesis via its interaction with LSD1.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.

Androgens stimulate erythropoiesis through the DNA-binding activity of the androgen receptor in non-hematopoietic cells.

BACKGROUND: Androgens function through DNA and non-DNA binding-dependent signalling of the androgen receptor (AR). How androgens promote erythropoiesis is not fully understood. DESIGN AND METHODS: To identify the androgen signalling pathway, we treated male mice lacking the second zinc finger of the DNA-binding domain of the AR (ARΔZF2 ) with non-aromatizable 5α-dihydrotestosterone (5α-DHT) or aromatizable testosterone. To distinguish direct hematopoietic and non-hematopoietic mechanisms, we performed bone marrow reconstitution experiments. RESULTS: In wild-type mice, 5α-DHT had greater erythroid activity than testosterone, which can be aromatized to estradiol. The erythroid response in wild-type mice following 5α-DHT treatment was associated with increased serum erythropoietin (EPO) and its downstream target erythroferrone, and hepcidin suppression. 5α-DHT had no erythroid activity in ARΔZF2 mice, proving the importance of DNA binding by the AR. Paradoxically, testosterone, but not 5α-DHT, suppressed EPO levels in ARΔZF2 mice, suggesting testosterone following aromatization may oppose the erythroid-stimulating effects of androgens. Female wild-type mice reconstituted with ARΔZF2 bone marrow cells remained responsive to 5α-DHT. In contrast, ARΔZF2 mice reconstituted with female wild-type bone marrow cells showed no response to 5α-DHT. CONCLUSION: Erythroid promoting effects of androgens are mediated through DNA binding-dependent actions of the AR in non-hematopoietic cells, including stimulating EPO expression.

STAT3-driven hematopoiesis and lymphopoiesis abnormalities are dependent on serine phosphorylation.

Deregulated activation of the latent transcription factor STAT3 has been implicated in the pathogenesis of myeloproliferative and lymphoproliferative hematologic disorders. The uncontrolled activation of STAT3 has traditionally been assigned to its elevated phosphorylation at tyrosine 705 (pY705) and associated nuclear transcriptional activity. By contrast, a transcriptional role for serine 727 phosphorylation (pS727) of STAT3 has recently emerged, suggesting that pS727 may account for the pathological activity of STAT3 in certain disease settings. Here, by coupling pS727-STAT3-deficient Stat3SA/SA mice with a STAT3-driven mouse model (gp130F/F) for myeloproliferative and lymphoproliferative pathologies, we reveal a key role for pS727-STAT3 in promoting multiple hematologic pathologies. The genetic blockade of pS727-STAT3 in gp130F/F:Stat3SA/SA mice ameliorated the neutrophilia, thrombocytosis, splenomegaly and lymphadenopathy that are features of gp130F/F mice. The protection against thrombocytosis in gp130F/F:Stat3SA/SA mice coincided with normalized megakaryopoiesis in both bone marrow and spleen compartments. Interestingly, pS727-STAT3-mediated abnormal lymphopoiesis in gp130F/F mice was more pronounced in lymph nodes compared to thymus, and was characterized by elevated numbers of B cells at the expense of T cells. Furthermore, pS727-STAT3 dependency for these hematologic pathologies coincided with transcriptional activity on STAT3-regulated genes, rather than its effect on mitochondrial and metabolic genes. Collectively, these findings suggest that pS727 plays a critical pathological role in modulating the transcriptional activity of STAT3 in hematologic disorders.

Hhex regulates murine lymphoid progenitor survival independently of Stat5 and Cdkn2a.

The transcription factor Hhex (hematopoietically expressed homeobox gene) is critical for development of multiple lymphoid lineages beyond the common lymphoid progenitor. In addition, Hhex regulates hematopoietic stem cell (HSC) self-renewal, emergency hematopoiesis, and acute myeloid leukemia initiation and maintenance. Hhex mediates its effects on HSCs and acute myeloid leukemia stem cells via repression of the Cdkn2a tumor suppressor locus. However, we report here that loss of Cdkn2a does not rescue the failure of lymphoid development caused by loss of Hhex. As loss of Hhex causes apoptosis of lymphoid progenitors associated with impaired Bcl2 expression and defective Stat5b signaling, we tested the effects of rescuing these pathways using transgenic mice. Expression of the anti-apoptotic factor Bcl2, but not activated Stat5, rescued the development of T-, B-, and NK-cell lineages in the absence of Hhex. These results indicate that Bcl2 expression, but not Stat5b signaling or loss of Cdkn2a, can overcome the lymphoid deficiencies caused by the absence of Hhex, suggesting that the primary role of this transcription factor is to promote survival of lymphoid progenitors during early lymphoid development.

PHF6 regulates hematopoietic stem and progenitor cells and its loss synergizes with expression of TLX3 to cause leukemia.

Somatically acquired mutations in PHF6 (plant homeodomain finger 6) frequently occur in hematopoietic malignancies and often coincide with ectopic expression of TLX3. However, there is no functional evidence to demonstrate whether these mutations contribute to tumorigenesis. Similarly, the role of PHF6 in hematopoiesis is unknown. We report here that Phf6 deletion in mice resulted in a reduced number of hematopoietic stem cells (HSCs), an increased number of hematopoietic progenitor cells, and an increased proportion of cycling stem and progenitor cells. Loss of PHF6 caused increased and sustained hematopoietic reconstitution in serial transplantation experiments. Interferon-stimulated gene expression was upregulated in the absence of PHF6 in hematopoietic stem and progenitor cells. The numbers of hematopoietic progenitor cells and cycling hematopoietic stem and progenitor cells were restored to normal by combined loss of PHF6 and the interferon α and ß receptor subunit 1. Ectopic expression of TLX3 alone caused partially penetrant leukemia. TLX3 expression and loss of PHF6 combined caused fully penetrant early-onset leukemia. Our data suggest that PHF6 is a hematopoietic tumor suppressor and is important for fine-tuning hematopoietic stem and progenitor cell homeostasis.

The NUP98-HOXD13 fusion oncogene induces thymocyte self-renewal via Lmo2/Lyl1.

T cell acute lymphoblastic leukaemia (T-ALL) cases include subfamilies that overexpress the TAL1/LMO, TLX1/3 and HOXA transcription factor oncogenes. While it has been shown that TAL1/LMO transcription factors induce self-renewal of thymocytes, whether this is true for other transcription factor oncogenes is unknown. To address this, we have studied NUP98-HOXD13-transgenic (NHD13-Tg) mice, which overexpress HOXA transcription factors throughout haematopoiesis and develop both myelodysplastic syndrome (MDS) progressing to acute myeloid leukaemia (AML) as well as T-ALL. We find that thymocytes from preleukaemic NHD13-Tg mice can serially transplant, demonstrating that they have self-renewal capacity. Transcriptome analysis shows that NHD13-Tg thymocytes exhibit a stem cell-like transcriptional programme closely resembling that induced by Lmo2, including Lmo2 itself and its critical cofactor Lyl1. To determine whether Lmo2/Lyl1 are required for NHD13-induced thymocyte self-renewal, NHD13-Tg mice were crossed with Lyl1 knockout mice. This showed that Lyl1 is essential for expression of the stem cell-like gene expression programme in thymocytes and self-renewal. Surprisingly however, NHD13 transgenic mice lacking Lyl1 showed accelerated T-ALL and absence of transformation to AML, associated with a loss of multipotent progenitors in the bone marrow. Thus multiple T cell oncogenes induce thymocyte self-renewal via Lmo2/Lyl1; however, NHD13 can also promote T-ALL via an alternative pathway.

Thymocyte self-renewal and oncogenic risk in immunodeficient mouse models: relevance for human gene therapy clinical trials targeting haematopoietic stem cell populations?

Emerging evidence indicates that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Here we discuss formative studies demonstrating that, in mice, early thymocytes acquire self-renewing potential when thymic progenitor supply is sub-physiological and the importance of cellular competition with this at-risk cell population to prevent lymphoid malignancy. We also consider the possibility that increased thymic residency time, established under conditions of limited cellular competition, may have contributed to oncogenesis observed in early SCID-X1 trials when combined with insertional activation of proto-oncogenes such as LMO2.

Hhex induces promyelocyte self-renewal and cooperates with growth factor independence to cause myeloid leukemia in mice.

The hematopoietically expressed homeobox (Hhex) transcription factor is overexpressed in human myeloid leukemias. Conditional knockout models of murine acute myeloid leukemia indicate that Hhex maintains leukemia stem cell self-renewal by enabling Polycomb-mediated epigenetic repression of the Cdkn2a tumor suppressor locus, encoding p16Ink4a and p19Arf However, whether Hhex overexpression also affects hematopoietic differentiation is unknown. To study this, we retrovirally overexpressed Hhex in hematopoietic progenitors. This enabled serial replating of myeloid progenitors, leading to the rapid establishment of interleukin-3 (IL-3)-dependent promyelocytic cell lines. Use of a Hhex-ERT2 fusion protein demonstrated that continuous nuclear Hhex is required for transformation, and structure function analysis demonstrated a requirement of the DNA-binding and N-terminal-repressive domains of Hhex for promyelocytic transformation. This included the N-terminal promyelocytic leukemia protein (Pml) interaction domain, although deletion of Pml failed to prevent Hhex-induced promyelocyte transformation, implying other critical partners. Furthermore, deletion of p16Ink4a or p19Arf did not promote promyelocyte transformation, indicating that repression of distinct Hhex target genes is required for this process. Indeed, transcriptome analysis showed that Hhex overexpression resulted in repression of several myeloid developmental genes. To test the potential for Hhex overexpression to contribute to leukemic transformation, Hhex-transformed promyelocyte lines were rendered growth factor-independent using a constitutively active IL-3 receptor common ß subunit (ßcV449E). The resultant cell lines resulted in a rapid promyelocytic leukemia in vivo. Thus, Hhex overexpression can contribute to myeloid leukemia via multiple mechanisms including differentiation blockade and enabling epigenetic repression of the Cdkn2a locus.

Analysis of Histone Modifications in Acute Myeloid Leukaemia Using Chromatin Immunoprecipitation.

Chromatin Immunoprecipitation (ChIP) using antibodies specific for histone modifications is a powerful technique for assessing the epigenetic states of cell populations by either quantitative PCR (ChIP-PCR) or next generation sequencing analysis (ChIP-Seq). Here we describe the procedure for ChIP of histone marks in myeloid leukaemia cell lines and the subsequent purification of genomic DNA associated with repressive and activating histone modifications for further analysis. This procedure can be widely applied to a variety of histone marks to assess both activating and repressive modifications in the context of myeloid leukaemia.

Hhex Regulates Hematopoietic Stem Cell Self-Renewal and Stress Hematopoiesis via Repression of Cdkn2a.

The hematopoietically expressed homeobox transcription factor (Hhex) is important for the maturation of definitive hematopoietic progenitors and B-cells during development. We have recently shown that in adult hematopoiesis, Hhex is dispensable for maintenance of hematopoietic stem cells (HSCs) and myeloid lineages but essential for the commitment of common lymphoid progenitors (CLPs) to lymphoid lineages. Here, we show that during serial bone marrow transplantation, Hhex-deleted HSCs are progressively lost, revealing an intrinsic defect in HSC self-renewal. Moreover, Hhex-deleted mice show markedly impaired hematopoietic recovery following myeloablation, due to a failure of progenitor expansion. In vitro, Hhex-null blast colonies were incapable of replating, implying a specific requirement for Hhex in immature progenitors. Transcriptome analysis of Hhex-null Lin- Sca+ Kit+ cells showed that Hhex deletion leads to derepression of polycomb repressive complex 2 (PRC2) and PRC1 target genes, including the Cdkn2a locus encoding the tumor suppressors p16Ink 4a and p19Arf . Indeed, loss of Cdkn2a restored the capacity of Hhex-null blast colonies to generate myeloid progenitors in vitro, as well as hematopoietic reconstitution following myeloablation in vivo. Thus, HSCs require Hhex to promote PRC2-mediated Cdkn2a repression to enable continued self-renewal and response to hematopoietic stress. Stem Cells 2017;35:1948-1957.