Increased CXCL10 (IP-10) is associated with advanced myeloproliferative neoplasms and its loss dampens erythrocytosis in mouse models.

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of the disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (tumor necrosis factor-alpha [TNF-α], interferon gamma-induced protein-10 [IP-10 or CXCL10]) and decreased survival (interleukin 8 [IL-8]). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2V617F/V617F TET2-/- double-mutant mice) compared with those with less severe phenotypes (e.g., CALRdel52 or JAK2+/V617F mice) and wild-type (WT) littermate controls. Although exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10-/- mice transplanted with disease-initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was noncell-autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10-/- mice into a series of MPN mouse models and showed that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together, these data point to a potential role for blocking IP-10 activity in the management of MPNs.

Convergent somatic evolution commences in utero in a germline ribosomopathy.

Clonal tracking of cells using somatic mutations permits exploration of clonal dynamics in human disease. Here, we perform whole genome sequencing of 323 haematopoietic colonies from 10 individuals with the inherited ribosomopathy Shwachman-Diamond syndrome to reconstruct haematopoietic phylogenies. In ~30% of colonies, we identify mutually exclusive mutations in TP53, EIF6, RPL5, RPL22, PRPF8, plus chromosome 7 and 15 aberrations that increase SBDS and EFL1 gene dosage, respectively. Target gene mutations commence in utero, resulting in a profusion of clonal expansions, with only a few haematopoietic stem cell lineages (mean 8, range 1-24) contributing ~50% of haematopoietic colonies across 8 individuals (range 4-100% clonality) by young adulthood. Rapid clonal expansion during disease transformation is associated with biallelic TP53 mutations and increased mutation burden. Our study highlights how convergent somatic mutation of the p53-dependent nucleolar surveillance pathway offsets the deleterious effects of germline ribosomopathy but increases opportunity for TP53-mutated cancer evolution.

[Detection of alcohol consumption at the first level of care in Mexico during the COVID-19 pandemicDetecção do consumo de álcool no nível da atenção primária no México durante a pandemia de COVID-19]. / Detección del consumo de alcohol en el primer nivel de atención en México durante la pandemia de COVID-19.

Objective: To measure the impact of the COVID-19 pandemic on the implementation of a program for timely detection, brief intervention, and referral for treatment of alcohol consumption in health centers at the first level of care in Mexico City. Methods: The data were collected at 18 health centers in Mexico City between 2019 and 2021, as part of a larger study. A total of 287 participating health professionals measured their patients' alcohol consumption using the AUDIT-C test. The patients' demographic aspects and consumption patterns were analyzed, as well as care actions (detection, brief intervention, provision of leaflets, monitoring, and referral) at three points in time: before the COVID-19 pandemic, during confinement, and after confinement. Parametric and nonparametric tests were performed to identify the relationship and differences between the variables at the three points. Results: A total of 9090 people who consumed alcohol were identified; detection of consumption fluctuated in the three periods studied (26%, 53%, and 37%, respectively). Medium- and high-risk consumption was more frequent among young men with higher schooling during and after confinement. In the post-confinement period, monitoring and referral actions increased. Conclusions: Measuring the impact of the pandemic on the implementation of a program for timely detection of alcohol consumption, brief intervention, and referral for treatment in health centers helps to develop health policies by age, gender, schooling, and type of consumption at the first level of care.

Objetivo: Medir o efeito da pandemia de COVID-19 na implementação de um programa de detecção oportuna, intervenção breve e encaminhamento para tratamento pelo consumo de álcool em centros de saúde no nível da atenção primária na Cidade do México. Métodos: Foram coletados dados em 18 centros de saúde na Cidade do México entre 2019 e 2021, como parte de um estudo mais amplo. Participaram 287 profissionais de saúde, que mediram o consumo de álcool de seus pacientes com o teste AUDIT-C. Foram analisados aspectos demográficos e o padrão de consumo dos pacientes, bem como as medidas em termos de atendimento (triagem, intervenção breve, entrega de folhetos, monitoramento e encaminhamento) em três momentos: antes da pandemia de COVID-19, durante o confinamento e após o confinamento. Foram feitos testes paramétricos e não paramétricos para identificar a relação e as diferenças entre as variáveis nos três períodos. Resultados: Foram identificadas 9.090 pessoas com consumo de álcool, sendo que a porcentagem de consumo detectada flutuou nos três períodos estudados (26%, 53% e 37%, respectivamente). O consumo de médio e alto risco foi mais prevalente entre homens jovens e com nível maior de escolaridade durante e após o confinamento. No período posterior ao confinamento, as medidas de monitoramento e encaminhamento aumentaram. Conclusões: Medir o impacto da pandemia na implementação de um programa de detecção oportuna, intervenção breve e encaminhamento para tratamento do consumo de álcool em centros de saúde ajuda na formulação de políticas de saúde por idade, gênero, escolaridade e tipo de consumo para o primeiro nível de atenção.

Psycho-Emotional Factors Associated with Depressive Symptoms during Lockdown Due to the COVID-19 Pandemic in the Mexican Population.

The COVID-19 pandemic has had a significant impact on mental health, leading to the increase of depressive symptoms. Identifying these symptoms and the factors associated with them in women and men will allow us to understand possible mechanisms of action and develop more specific interventions. An online survey was conducted from 1 May to 30 June 2020 using snowball sampling; the final sample comprised 4122 adult inhabitants of Mexico; 35% of the total sample displayed moderate-to-severe depressive symptoms, with a greater proportion of depression being among female respondents. A logistic regression analysis revealed that individuals under 30 years of age, those with high levels of stress due to social distancing, those with negative emotions, and those who reported a significant impact of the pandemic on their lives have a higher risk of depression. Women with a history of mental health treatment and men with a history of chronic disease were also more likely to experience depressive symptoms. Social environment and sex are factors that intervene in the development of depressive symptoms, meaning that appropriate early identification and intervention models should be designed for the care of men and women in highly disruptive situations such as the recent pandemic.

Detección del consumo de alcohol en el primer nivel de atención en México durante la pandemia de COVID-19 / Detection of alcohol consumption at the first level of care in Mexico during the COVID-19 pandemic / Detecção do consumo de álcool no nível da atenção primária no México durante a pandemia de COVID-19

RESUMEN Objetivo. Medir el efecto de la pandemia de COVID-19 en la implementación de un programa de detección oportuna, intervención breve y referencia para tratamiento por consumo de alcohol en centros de salud del primer nivel de atención de la Ciudad de México. Métodos. Se recopilaron los datos en 18 centros de salud de la Ciudad de México entre 2019 y 2021, como parte de un estudio más amplio. Participaron 287 profesionales de la salud, quienes midieron el consumo de alcohol de sus pacientes con la prueba AUDIT-C. Se analizaron aspectos demográficos y patrones de consumo de los pacientes, así como las acciones de atención (detección, intervención breve, entrega de folletos, monitoreo y referencia) en tres momentos: antes de la pandemia de COVID-19, durante el confinamiento y luego del confinamiento. Se realizaron pruebas paramétricas y no paramétricas para identificar la relación y las diferencias entre las variables en los tres períodos. Resultados. Se identificaron 9 090 personas con consumo de alcohol; el porcentaje de detección del consumo tuvo fluctuaciones en los tres períodos estudiados (26%, 53% y 37%, respectivamente). El consumo de riesgo medio y alto fue más frecuente entre hombres jóvenes y con mayor escolaridad durante y después del confinamiento. En el período posconfinamiento aumentaron las acciones de monitoreo y referencia. Conclusiones. La medición del impacto de la pandemia en la implementación de un programa de detección oportuna, asesoramiento breve y referencia para el tratamiento del consumo de alcohol en centros de salud ayuda a elaborar políticas en salud por edad, género, escolaridad y tipo de consumo para el primer nivel de atención.

ABSTRACT Objective. To measure the impact of the COVID-19 pandemic on the implementation of a program for timely detection, brief intervention, and referral for treatment of alcohol consumption in health centers at the first level of care in Mexico City. Methods. The data were collected at 18 health centers in Mexico City between 2019 and 2021, as part of a larger study. A total of 287 participating health professionals measured their patients' alcohol consumption using the AUDIT-C test. The patients' demographic aspects and consumption patterns were analyzed, as well as care actions (detection, brief intervention, provision of leaflets, monitoring, and referral) at three points in time: before the COVID-19 pandemic, during confinement, and after confinement. Parametric and nonparametric tests were performed to identify the relationship and differences between the variables at the three points. Results. A total of 9090 people who consumed alcohol were identified; detection of consumption fluctuated in the three periods studied (26%, 53%, and 37%, respectively). Medium- and high-risk consumption was more frequent among young men with higher schooling during and after confinement. In the post-confinement period, monitoring and referral actions increased. Conclusions. Measuring the impact of the pandemic on the implementation of a program for timely detection of alcohol consumption, brief intervention, and referral for treatment in health centers helps to develop health policies by age, gender, schooling, and type of consumption at the first level of care.

RESUMO Objetivo. Medir o efeito da pandemia de COVID-19 na implementação de um programa de detecção oportuna, intervenção breve e encaminhamento para tratamento pelo consumo de álcool em centros de saúde no nível da atenção primária na Cidade do México. Métodos. Foram coletados dados em 18 centros de saúde na Cidade do México entre 2019 e 2021, como parte de um estudo mais amplo. Participaram 287 profissionais de saúde, que mediram o consumo de álcool de seus pacientes com o teste AUDIT-C. Foram analisados aspectos demográficos e o padrão de consumo dos pacientes, bem como as medidas em termos de atendimento (triagem, intervenção breve, entrega de folhetos, monitoramento e encaminhamento) em três momentos: antes da pandemia de COVID-19, durante o confinamento e após o confinamento. Foram feitos testes paramétricos e não paramétricos para identificar a relação e as diferenças entre as variáveis nos três períodos. Resultados. Foram identificadas 9.090 pessoas com consumo de álcool, sendo que a porcentagem de consumo detectada flutuou nos três períodos estudados (26%, 53% e 37%, respectivamente). O consumo de médio e alto risco foi mais prevalente entre homens jovens e com nível maior de escolaridade durante e após o confinamento. No período posterior ao confinamento, as medidas de monitoramento e encaminhamento aumentaram. Conclusões. Medir o impacto da pandemia na implementação de um programa de detecção oportuna, intervenção breve e encaminhamento para tratamento do consumo de álcool em centros de saúde ajuda na formulação de políticas de saúde por idade, gênero, escolaridade e tipo de consumo para o primeiro nível de atenção.

Diverse mutational landscapes in human lymphocytes.

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

Identification and characterization of in vitro expanded hematopoietic stem cells.

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200-fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non-HSCs and single cell-initiated cultures have substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. By directly linking single-clone functional transplantation data with single-clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Humor in Life Scale (EHV): Development and Evidence of Validity and Reliability in Spanish Speakers / Escala de Humor ante la Vida (EHV): Desarrollo, Evidencias de Validez y Confiabilidad en Hispanohablantes

Abstract Despite the growing attention given to the study of humor, a measure in the Spanish language of people's experience and enjoyment of humor in everyday life is still missing. The present study describes the development and validation of the EHV (from the Spanish Escala de Humor ante la Vida, Humor in Life Scale). In phase I, the items were developed using focus groups and interviews; their content validity was assessed through expert judgment. In phase II, the EHV was answered by two Mexican samples (N=1380), women and men, from 18 to 66 years; it was administered together with the Numeric Rating Scale of Humor and the Positive and Negative Affect Schedule, in both printed and electronic formats. The exploratory factor analysis supported the single factor structure and the confirmatory factor analysis showed adequate fit indices for the final eight-item scale; the factor structure was partially invariant between sexes. Reliability indices were satisfactory. Convergent and discriminant validity tests showed that the EHV is related to a global measure of humor and positive and negative affect. In sum, the results indicate that the EHV is a brief, valid and reliable measure to assess humor in life in Spanish speakers.

Resumen A pesar de la creciente atención que se ha otorgado al estudio del sentido del humor, no se contaba con un instrumento en español que evaluara la experiencia y el disfrute del humor en la vida diaria. La investigación constó de dos fases. En la primera, a partir de grupos focales y entrevistas, se elaboraron 30 reactivos potenciales. Para evaluar su validez de contenido, se obtuvieron índices V de Aiken de los juicios de cuatro expertos. Los 14 reactivos que alcanzaron el criterio fueron sometidos a un piloteo, después del cual la escala quedó conformada por 11 reactivos, con siete opciones de respuesta. En la segunda fase, la EHV se aplicó, junto con la Escala de Evaluación Numérica del Humor (una medida global de humor) y la Escala de Afecto Positivo y Negativo (PANAS), a dos muestras de población general (n1=1380 y n1=550), hombres y mujeres con edades de 18 a 66 años. La aplicación de la batería se efectuó tanto en línea como en formato impreso en diversos lugares públicos. El análisis factorial exploratorio mostró una estructura unidimensional que explicó el 55.97% de la varianza total; dos reactivos fueron eliminados. Se obtuvieron índices de ajuste adecuados al someter a análisis factorial confirmatorio a la EHV, los cuales mejoraron con la eliminación de un reactivo, por lo que la escala quedó finalmente conformada por ocho reactivos. El AVE fue >.50 y los índices de confiabilidad alfa y omega resultaron >.91. Al evaluar la invarianza de la estructura factorial de la EHV por sexo, ésta resultó parcial, ya que se obtuvieron índices dentro de los criterios señalados para el modelo métrico y para el fuerte, pero no todos para el estricto. La correlación entre la EHV y la Escala de Evaluación Numérica del Humor resultó, como se esperaba, alta y positiva, así como con el factor de afecto positivo del PANAS, en tanto que lo opuesto se observó con el factor de afecto negativo. Tener creencias espirituales o religiosas y tener una pareja no mostraron relación con los puntajes de la EHV. Estos resultaron apoyaron la validez convergente y discriminante de la EHV. En conclusión, la EHV es un instrumento breve y unidimensional, con evidencias de validez y confiabilidad, que evalúa la experiencia y disfrute del humor en la vida cotidiana en personas de habla hispana.

Development and Psychometric Properties of the Adversity and Stress Scale (ASS): Validation in the Adult Mexican Population.

Since stress is known to play a role in the development of physical and mental illness, empirically validated measurements are required to assess the effect of adverse events such as the COVID-19 pandemic. The purpose of this study was to develop and evaluate the psychometric properties of the Adversity and Stress Scale (ASS). A sample of 3937 adults living in Mexico was used. The structure of the instrument was evaluated using exploratory factor analysis (EFA) and confirmatory factor analysis (CFA). Construct validity was measured through associations between the ASS and psychological symptoms. In the EFA, the relational and contextual dimensions of stress were identified. A good fit was obtained in the CFA (CFI = 0.980, RMSEA = 0.040). The ASS score was associated with all the selected variables in the expected direction, and internal consistency was α = .86. The ASS is a valid, reliable measure, with the potential to be used in other adverse events.

Protocol to maintain single functional mouse hematopoietic stem cells in vitro without cell division.

This protocol details the isolation and in vitro maintenance of single hematopoietic stem cells (HSCs) in the absence of the bone marrow niche. The HSCs do not divide over 7 days and fully retain their long-term functional capacity in transplantation assays. Following hibernation culture, HSCs can be used to study quiescence exit and can be genetically manipulated as single cells prior to division. For complete details on the use and execution of this protocol, please refer to Oedekoven et al. (2021).

Psychoactive Substance Use and Its Relationship to Stress, Emotional State, Depressive Symptomatology, and Perceived Threat During the COVID-19 Pandemic in Mexico.

People can increase their use of psychoactive substances in response to stressful situations as a maladaptive mechanism for reducing negative affective states. It is therefore necessary to examine changes in the use of such substances and their relationship to mental health in light of the COVID-19 pandemic. Objective: Evaluate the relationship between psychoactive substances and stress, emotional state, and symptomatology during the COVID-19 lockdown in Mexico. Method: A national survey was conducted, using the free Google Forms platform, of residents of Mexico aged 18 and older. The survey was disseminated through social media. Results: The sample comprised 4,122 individuals, mostly women (71.8%), with an age range of 18-81 years (M = 37.08, SD = 12.689), of which 46.8% were single, and 42.9% married. In general, there was a reduction in substance use during the first 2 months of the quarantine; the most commonly used substances were alcohol, tobacco, and tranquilizers. Respondents who described having greater use than before the pandemic presented greater stress, depressive symptomatology, and perceived threat than those who did not use substances. Conclusions: Respondents who did not use substances reported lower levels of stress, depressive symptomatology, impact of the coronavirus pandemic, and perception of its threat. Women reported greater stress, depressive symptomatology, and emotional intensity than men.

Hematopoietic stem cells retain functional potential and molecular identity in hibernation cultures.

Advances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs.

Longitudinal Cytokine Profiling Identifies GRO-α and EGF as Potential Biomarkers of Disease Progression in Essential Thrombocythemia.

Myeloproliferative neoplasms (MPNs) are characterized by deregulation of mature blood cell production and increased risk of myelofibrosis (MF) and leukemic transformation. Numerous driver mutations have been identified but substantial disease heterogeneity remains unexplained, implying the involvement of additional as yet unidentified factors. The inflammatory microenvironment has recently attracted attention as a crucial factor in MPN biology, in particular whether inflammatory cytokines and chemokines contribute to disease establishment or progression. Here we present a large-scale study of serum cytokine profiles in more than 400 MPN patients and identify an essential thrombocythemia (ET)-specific inflammatory cytokine signature consisting of Eotaxin, GRO-α, and EGF. Levels of 2 of these markers (GRO-α and EGF) in ET patients were associated with disease transformation in initial sample collection (GRO-α) or longitudinal sampling (EGF). In ET patients with extensive genomic profiling data (n = 183) cytokine levels added significant prognostic value for predicting transformation from ET to MF. Furthermore, CD56+CD14+ pro-inflammatory monocytes were identified as a novel source of increased GRO-α levels. These data implicate the immune cell microenvironment as a significant player in ET disease evolution and illustrate the utility of cytokines as potential biomarkers for reaching beyond genomic classification for disease stratification and monitoring.

Population dynamics of normal human blood inferred from somatic mutations.

Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000-200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans.

Single-cell approaches identify the molecular network driving malignant hematopoietic stem cell self-renewal.

Recent advances in single-cell technologies have permitted the investigation of heterogeneous cell populations at previously unattainable resolution. Here we apply such approaches to resolve the molecular mechanisms driving disease in mouse hematopoietic stem cells (HSCs), using JAK2V617F mutant myeloproliferative neoplasms (MPNs) as a model. Single-cell gene expression and functional assays identified a subset of JAK2V617F mutant HSCs that display defective self-renewal. This defect is rescued at the single HSC level by crossing JAK2V617F mice with mice lacking TET2, the most commonly comutated gene in patients with MPN. Single-cell gene expression profiling of JAK2V617F-mutant HSCs revealed a loss of specific regulator genes, some of which were restored to normal levels in single TET2/JAK2 mutant HSCs. Of these, Bmi1 and, to a lesser extent, Pbx1 and Meis1 overexpression in JAK2-mutant HSCs could drive a disease phenotype and retain durable stem cell self-renewal in functional assays. Together, these single-cell approaches refine the molecules involved in clonal expansion of MPNs and have broad implications for deconstructing the molecular network of normal and malignant stem cells.

Leukemia stem cells in T-ALL require active Hif1α and Wnt signaling.

The Wnt signaling pathway has been shown to play important roles in normal hematopoietic stem cell biology and in the development of both acute and chronic myelogenous leukemia. Its role in maintaining established leukemia stem cells, which are more directly relevant to patients with disease, however, is less clear. To address what role Wnt signaling may play in T-cell acute lymphoblastic leukemia (T-ALL), we used a stably integrated fluorescent Wnt reporter construct to interrogate endogenous Wnt signaling activity in vivo. In this study, we report that active Wnt signaling is restricted to minor subpopulations within bulk tumors, that these Wnt-active subsets are highly enriched for leukemia-initiating cells (LICs), and that genetic inactivation of ß-catenin severely reduces LIC frequency. We show further that ß-catenin transcription is upregulated by hypoxia through hypoxia-inducible factor 1α (Hif1α) stabilization, and that deletion of Hif1α also severely reduces LIC frequency. Of note, the deletion of ß-catenin or Hif1α did not impair the growth or viability of bulk tumor cells, suggesting that elements of the Wnt and Hif pathways specifically support leukemia stem cells. We also confirm the relevance of these findings to human disease using cell lines and patient-derived xenografts, suggesting that targeting these pathways could benefit patients with T-ALL.