Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia.

While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.

Not All Created Equal: Lineage Hard-Wiring in the Production of Blood.

The multiple cell types comprising blood have been thought to emerge from progenitors with progressively narrower lineage options. New data suggest that lineage fate may be determined earlier than thought and that myeloid progenitor populations are aggregates of individual lineage-restricted cells.

Xist RNA is a potent suppressor of hematologic cancer in mice.

X chromosome aneuploidies have long been associated with human cancers, but causality has not been established. In mammals, X chromosome inactivation (XCI) is triggered by Xist RNA to equalize gene expression between the sexes. Here we delete Xist in the blood compartment of mice and demonstrate that mutant females develop a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome (mixed MPN/MDS) with 100% penetrance. Significant disease components include primary myelofibrosis, leukemia, histiocytic sarcoma, and vasculitis. Xist-deficient hematopoietic stem cells (HSCs) show aberrant maturation and age-dependent loss. Reconstitution experiments indicate that MPN/MDS and myelofibrosis are of hematopoietic rather than stromal origin. We propose that Xist loss results in X reactivation and consequent genome-wide changes that lead to cancer, thereby causally linking the X chromosome to cancer in mice. Thus, Xist RNA not only is required to maintain XCI but also suppresses cancer in vivo.

Proton export alkalinizes intracellular pH and reprograms carbon metabolism to drive normal and malignant cell growth.

Proton export is often considered a detoxifying process in animal cells, with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH, thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels, NADPH/NADP+ ratio, and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy, where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth.

CRISPR-Cas9 system: a novel and promising era of genotherapy for beta-hemoglobinopathies, hematological malignancy, and hemophilia.

Gene therapy represents a significant potential to revolutionize the field of hematology with applications in correcting genetic mutations, generating cell lines and animal models, and improving the feasibility and efficacy of cancer immunotherapy. Compared to different genetic engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) emerged as an effective and versatile genetic editor with the ability to precisely modify the genome. The applications of genetic engineering in various hematological disorders have shown encouraging results. Monogenic hematological disorders can conceivably be corrected with single gene modification. Through the use of CRISPR-CAS9, restoration of functional red blood cells and hemostasis factors were successfully attained in sickle cell anemia, beta-thalassemia, and hemophilia disorders. Our understanding of hemato-oncology has been advanced via CRIPSR-CAS9 technology. CRISPR-CAS9 aided to build a platform of mutated genes responsible for cell survival and proliferation in leukemia. Therapeutic application of CRISPR-CAS9 when combined with chimeric antigen receptor (CAR) T cell therapy in multiple myeloma and acute lymphoblastic leukemia was feasible with attenuation of CAR T cell therapy pitfalls. Our review outlines the latest literature on the utilization of CRISPR-Cas9 in the treatment of beta-hemoglobinopathies and hemophilia disorders. We present the strategies that were employed and the findings of preclinical and clinical trials. Also, the review will discuss gene engineering in the field of hemato-oncology as a proper tool to facilitate and overcome the drawbacks of chimeric antigen receptor T cell therapy (CAR-T).

Metabolomic Study Using Time-of-Flight Mass Spectrometry Reveals Novel Urinary Biomarkers for Gaucher Disease Type 1.

Gaucher disease (GD) is a lysosomal storage disorder resulting from a biallelic mutation in the gene GBA1, leading to deficiencies in the enzyme ß-glucocerebrosidase (Gcase). Inabilities of the Gcase to catabolize its substrate result in the accumulation of sphingolipids in macrophages, which impairs the cell functions and ultimately leads to multisystemic clinical manifestations. Important variability in symptoms and manifestations may lead to challenging diagnosis and patient care. Plasma glucosylsphingosine (lyso-Gb1) is a biomarker frequently used for prognosis, monitoring, and patient follow-up. While lyso-Gb1 appears to be a valid biomarker, few studies have investigated other matrices for potential GD biomarkers. The main objective of this study was to investigate the urine matrix as a potential source of new GD biomarkers by performing a metabolomic study using time-of-flight mass spectrometry. Our study highlighted a significant increase of eight urinary lyso-Gb1 analogues. Moreover, a novel class of biomarkers, named polycyclic lyso-Gb1 analogues, was identified. These four new molecules were more elevated than lyso-Gb1 and related analogues in urine specimens of GD patients. Further investigations are warranted to validate the efficiency of these newly found biomarkers on a larger cohort of Gaucher patients and to compare them with plasma biomarkers currently quantified in clinical laboratories.

Identification of a Reliable Biomarker Profile for the Diagnosis of Gaucher Disease Type 1 Patients Using a Mass Spectrometry-Based Metabolomic Approach.

Gaucher disease (GD) is a rare autosomal recessive multisystemic lysosomal storage disorder presenting a marked phenotypic and genotypic variability. GD is caused by a deficiency in the glucocerebrosidase enzyme. The diagnosis of GD remains challenging because of the large clinical spectrum associated with the disease. Moreover, GD biomarkers are often not sensitive enough and can be subject to polymorphic variations. The main objective of this study was to perform a metabolomic study using an ultra-performance liquid chromatography system coupled to a time-of-flight mass spectrometer to identify novel GD biomarkers. Following the analysis of plasma samples from patients with GD, and age- and gender-matched control samples, supervised statistical analyses were used to find the best molecules to differentiate the two groups. Targeted biomarkers were structurally elucidated using accurate mass measurements and tandem mass spectrometry. This metabolomic study was successful in highlighting seven biomarkers associated with GD. Fragmentation tests revealed that these latter biomarkers were lyso-Gb1 (glucosylsphingosine) and four related analogs (with the following modifications on the sphingosine moiety: -C2H4, -H2, -H2+O, and +H2O), sphingosylphosphorylcholine, and N-palmitoyl-O-phosphocholineserine. Based on the plasma biomarker distribution, we suggest the evaluation of this GD biomarker profile, which might facilitate early diagnosis, monitoring, and follow-up of patients.

ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells.

The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521/Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521-deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.

Nucleic acid therapeutics as differentiation agents for myeloid leukemias.

Differentiation therapy has proven to be a success story for patients with acute promyelocytic leukemia. However, the remaining subtypes of acute myeloid leukemia (AML) are treated with cytotoxic chemotherapies that have limited efficacy and a high likelihood of resistance. As differentiation arrest is a hallmark of AML, there is increased interest in developing differentiation-inducing agents to enhance disease-free survival. Here, we provide a comprehensive review of current reports and future avenues of nucleic acid therapeutics for AML, focusing on the use of targeted nucleic acid drugs to promote differentiation. Specifically, we compare and discuss the precision of small interfering RNA, small activating RNA, antisense oligonucleotides, and aptamers to modulate gene expression patterns that drive leukemic cell differentiation. We delve into preclinical and clinical studies that demonstrate the efficacy of nucleic acid-based differentiation therapies to induce leukemic cell maturation and reduce disease burden. By directly influencing the expression of key genes involved in myeloid maturation, nucleic acid therapeutics hold the potential to induce the differentiation of leukemic cells towards a more mature and less aggressive phenotype. Furthermore, we discuss the most critical challenges associated with developing nucleic acid therapeutics for myeloid malignancies. By introducing the progress in the field and identifying future opportunities, we aim to highlight the power of nucleic acid therapeutics in reshaping the landscape of myeloid leukemia treatment.

Leukemia aggressiveness is driven by chromatin remodeling and expression changes of core regulators.

Molecular mechanisms driving clonal aggressiveness in leukemia are not fully understood. We tracked and analyzed two mouse MLL-rearranged leukemic clones independently evolving towards higher aggressiveness. More aggressive subclones lost their growth differential ex vivo but restored it upon secondary transplantation, suggesting molecular memory of aggressiveness. Development of aggressiveness was associated with clone-specific gradual modulation of chromatin states and expression levels across the genome, with a surprising preferential trend of reversing the earlier changes between normal and leukemic progenitors. To focus on the core aggressiveness program, we identified genes with consistent changes of expression and chromatin marks that were maintained in vivo and ex vivo in both clones. Overexpressing selected core genes (Smad1 as aggressiveness driver, Irx5 and Plag1 as suppressors) affected leukemic progenitor growth in the predicted way and had convergent downstream effects on central transcription factors and repressive epigenetic modifiers, suggesting a broader regulatory network of leukemic aggressiveness.

Trained immunity and epigenetic memory in long-term self-renewing hematopoietic cells.

Immunologic memory is a feature typically ascribed to the adaptive arm of the immune system. However, recent studies have demonstrated that hematopoietic stem cells (HSCs) and innate immune cells such as monocytes and macrophages can gain epigenetic signatures to enhance their response in the context of reinfection. This suggests the presence of long-term memory, a phenomenon referred to as trained immunity. Trained immunity in HSCs can occur via changes in the epigenetic landscape and enhanced chromatin accessibility in lineage-specific genes, as well as through metabolic alterations. These changes can lead to a skewing in lineage bias, particularly enhanced myelopoiesis and the generation of epigenetically modified innate immune cells that provide better protection against pathogens on secondary infection. Here, we summarize recent advancements in trained immunity and epigenetic memory formation in HSCs and self-renewing alveolar macrophages, which was the focus of the Spring 2022 International Society for Experimental Hematology (ISEH) webinar.

PARP-1 improves leukemia outcomes by inducing parthanatos during chemotherapy.

Previous chemotherapy research has focused almost exclusively on apoptosis. Here, a standard frontline drug combination of cytarabine and idarubicin induces distinct features of caspase-independent, poly(ADP-ribose) polymerase 1 (PARP-1)-mediated programmed cell death "parthanatos" in acute myeloid leukemia (AML) cell lines (n = 3/10 tested), peripheral blood mononuclear cells from healthy human donors (n = 10/10 tested), and primary cell samples from patients with AML (n = 18/39 tested, French-American-British subtypes M4 and M5). A 3-fold improvement in survival rates is observed in the parthanatos-positive versus -negative patient groups (hazard ratio [HR] = 0.28-0.37, p = 0.002-0.046). Manipulation of PARP-1 activity in parthanatos-competent cells reveals higher drug sensitivity in cells that have basal PARP-1 levels as compared with those subjected to PARP-1 overexpression or suppression. The same trends are observed in RNA expression databases and support the conclusion that PARP-1 can have optimal levels for favorable chemotherapeutic responses.

CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax.

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both in vitro and in vivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

Quantitation of a Urinary Profile of Biomarkers in Gaucher Disease Type 1 Patients Using Tandem Mass Spectrometry.

Gaucher disease is a rare inherited disorder caused by a deficiency of the lysosomal acid beta-glucocerebrosidase enzyme. Metabolomic studies by our group targeted several new potential urinary biomarkers. Apart from lyso-Gb1, these studies highlighted lyso-Gb1 analogs -28, -26, -12 (A/B), +2, +14, +16 (A/B), +30, and +32 Da, and polycyclic lyso-Gb1 analogs 362, 366, 390, and 394 Da. The main objective of the current study was to develop and validate a robust UPLC-MS/MS method to study the urine distribution of these biomarkers in patients. METHOD: Urine samples were purified using solid-phase extraction. A 12 min UPLC-MS/MS method was developed. RESULTS: Validation assays revealed high precision and accuracy for creatinine and lyso-Gb1. Most lyso-Gb1 analogs had good recovery rates and high intra- and interday precision assays. Biomarker-estimated LOD and LOQ levels ranged from 56-109 pM to 186-354 pM, respectively. Comparison between GD patients and healthy controls showed significant differences in most biomarker levels. Typically, treated GD patients presented lower biomarker levels compared to untreated patients. CONCLUSIONS: These data suggest that the metabolites investigated might be interesting GD biomarkers. More studies with a larger cohort of patients will be needed to better understand the clinical significance of these GD biomarkers.

Recent Advances in Cancer Drug Discovery Through the Use of Phenotypic Reporter Systems, Connectivity Mapping, and Pooled CRISPR Screening.

Multi-omic approaches offer an unprecedented overview of the development, plasticity, and resistance of cancer. However, the translation from anti-cancer compounds identified in vitro to clinically active drugs have a notoriously low success rate. Here, we review how technical advances in cell culture, robotics, computational biology, and development of reporter systems have transformed drug discovery, enabling screening approaches tailored to clinically relevant functional readouts (e.g., bypassing drug resistance). Illustrating with selected examples of "success stories," we describe the process of phenotype-based high-throughput drug screening to target malignant cells or the immune system. Second, we describe computational approaches that link transcriptomic profiling of cancers with existing pharmaceutical compounds to accelerate drug repurposing. Finally, we review how CRISPR-based screening can be applied for the discovery of mechanisms of drug resistance and sensitization. Overall, we explore how the complementary strengths of each of these approaches allow them to transform the paradigm of pre-clinical drug development.

m6A RNA modifications: Key regulators of normal and malignant hematopoiesis.

Post-transcriptional RNA modifications determine RNA fate by influencing numerous processes such as translation, decay and localization. One of the most abundant RNA modifications is N6-methyladenoside (m6A), which has been shown to be important in healthy as well as malignant hematopoiesis. Several proteins representing key players in m6A RNA biology, such as m6A writers, erasers and readers, were recently reported to be essential for hematopoietic stem cell (HSC) function. In leukemia, expression of m6A regulators has been shown to be increased, opening up potential opportunities for therapeutic exploitation by targeting them in blood malignancies. These recent discoveries were the focus of the Fall 2021 International Society for Experimental Hematology New Investigators webinar. We review here the latest findings in the field of mRNA modifications in normal and malignant hematopoiesis and how this might open up novel therapeutic options.

Quantitation of a plasma biomarker profile for the early detection of Gaucher disease type 1 patients.

Aim: Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid ß-glucocerebrosidase. Recent metabolomic studies highlighted several new metabolites increased in the plasma of GD patients. We aimed to develop and validate a UPLC-MS/MS method allowing a relative quantitation of lyso-Gb1 and lyso-Gb1 analogs -28, -12, -2, +14, +16 and +18 Da in addition to sphingosylphosphorylcholine, N-palmitoyl-O-phosphocholine to study potential correlations with clinical manifestations. Methodology & results: Following solid-phase extraction, plasma samples were evaporated and resuspended in 100 µl of resuspension solution. Three microliter is injected into the UPLC-MS/MS for analysis. Conclusion: All biomarkers studied were increased in GD patients. Significant correlations were observed between specific analogs and hematological, and visceral complications, as well as overall disease severity.

In vivo genome-wide CRISPR screening in murine acute myeloid leukemia uncovers microenvironmental dependencies.

Genome-wide CRISPR screens have been extremely useful in identifying therapeutic targets in diverse cancers by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in 2 in vivo murine models of acute myeloid leukemia (AML) driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the major histocompatibility complex class I complex, Cd47, complement receptor Cr1l, and the ß-4-galactosylation pathway. Importantly, several of these in vivo-specific hits have a prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as having in vivo-specific dependency with high prognostic relevance. Overall, we show an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.

Lysosphingolipid urine screening test using mass spectrometry for the early detection of lysosomal storage disorders.

Background: Sphingolipidoses are caused by a defective sphingolipid catabolism, leading to an accumulation of several glycolipid species in tissues and resulting in neurotoxicity and severe systemic manifestations. Methods & results: Urine samples from controls and patients were purified by solid-phase extraction prior to the analysis by ultra-high-performance liquid chromatography (UPLC) combined with MS/MS. A UPLC-MS/MS method for the analysis of 21 urinary creatinine-normalized biomarkers for eight diseases was developed and validated. Conclusion: Considering the growing demand to identify patients with different sphingolipidoses early and reliably, this methodology will be applied for high-risk screening to target efficiently patients with various sphingolipidoses.