Phase 3 randomized trial of mavorixafor, CXCR4 antagonist, in WHIM syndrome.

We investigated efficacy and safety of mavorixafor, an oral CXCR4 antagonist for participants with Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome, a rare immunodeficiency caused by CXCR4 gain-of-function variants. This randomized (1:1), double-blind, placebo-controlled, phase 3 trial enrolled participants aged ≥12 years with WHIM syndrome and absolute neutrophil count (ANC) ≤400/µL. Participants received once-daily mavorixafor or placebo for 52 weeks. Primary endpoint was time (hours) above ANC threshold ≥500/µL (TATANC; over 24 hours). Secondary endpoints included TAT absolute lymphocyte count ≥1000/µL (TATALC; defined similar to TATANC); absolute changes in white blood cell (WBC), ANC, and ALC from baseline; annualized infection rate; infection duration and total infection score (combined infection number/severity). In 31 participants (mavorixafor, n=14; placebo, n=17), mavorixafor least squares (LS) mean TATANC was 15.0 hours, placebo 2.8 hours (P<0.001). Mavorixafor LS mean TATALC was 15.8 hours, placebo 4.6 hours (P<0.001). Higher absolute WBC, ANC, and ALC levels were seen with mavorixafor than placebo at each timepoint assessed. Annualized infection rates were 60% lower with mavorixafor versus placebo (LS mean 1.7 versus 4.2; nominal P=0.007) and total infection scores were 40% lower (7.4 [95% CI, 1.6-13.2] versus 12.3 [95% CI, 7.2-17.3]). Treatment with mavorixafor reduced infection frequency, severity, duration, and antibiotic use. No discontinuations occurred due to treatment-emergent adverse events (TEAEs); no related serious TEAEs were observed. Overall, mavorixafor-treated participants showed significant increases in LS mean TATANC and TATALC, reduced infection frequency, severity/duration. Mavorixafor was well tolerated in participants with WHIM syndrome. Trial was registered at ClinicalTrials.gov NCT03995108.

T/myeloid mixed phenotype acute leukaemia harbouring TLX3::BCL11B with TLX3 activation.

T/myeloid mixed phenotype acute leukaemia (MPAL) is a rare aggressive acute leukaemia with poorly understood pathogenesis. Herein, we report two cases of T/myeloid MPAL harbouring BCL11B-associated structural variants that activate TLX3 (TLX3::BCL11B-TLX3-activation) by genome sequencing and transcriptomic analyses. Both patients were young males with extramedullary involvement. Cooperative gene alterations characteristic of T/myeloid MPAL and T-lymphoblastic leukaemia (T-ALL) were detected. Both patients achieved initial remission following lineage-matched ALL-based therapy with one patient requiring a lineage-switched myeloid-based therapy. Our study is the first to demonstrate the clinicopathological and genomic features of TLX3::BCL11B-TLX3-activated T/myeloid MPAL and provide insights into leukaemogenesis.

Structural variation cooperates with permissive chromatin to control enhancer hijacking-mediated oncogenic transcription.

Structural variants (SVs) involving enhancer hijacking can rewire chromatin topologies to cause oncogene activation in human cancers, including hematologic malignancies; however, because of the lack of tools to assess their effects on gene regulation and chromatin organization, the molecular determinants for the functional output of enhancer hijacking remain poorly understood. Here, we developed a multimodal approach to integrate genome sequencing, chromosome conformation, chromatin state, and transcriptomic alteration for quantitative analysis of transcriptional effects and structural reorganization imposed by SVs in leukemic genomes. We identified known and new pathogenic SVs, including recurrent t(5;14) translocations that cause the hijacking of BCL11B enhancers for the allele-specific activation of TLX3 in a subtype of pediatric leukemia. Epigenetic perturbation of SV-hijacked BCL11B enhancers impairs TLX3 transcription, which are required for the growth of t(5;14) leukemia cells. By CRISPR engineering of patient-derived t(5;14) in isogenic leukemia cells, we uncovered a new mechanism whereby the transcriptional output of SV-induced BCL11B enhancer hijacking is dependent on the loss of DNA hypermethylation at the TLX3 promoter. Our results highlight the importance of the cooperation between genetic alteration and permissive chromatin as a critical determinant of SV-mediated oncogene activation, with implications for understanding aberrant gene transcription after epigenetic therapies in patients with leukemia. Hence, leveraging the interdependency of genetic alteration on chromatin variation may provide new opportunities to reprogram gene regulation as targeted interventions in human disease.

Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance.

Mutations in IDH genes occur frequently in acute myeloid leukemia (AML) and other human cancers to generate the oncometabolite R-2HG. Allosteric inhibition of mutant IDH suppresses R-2HG production in a subset of patients with AML; however, acquired resistance emerges as a new challenge, and the underlying mechanisms remain incompletely understood. Here we establish isogenic leukemia cells containing common IDH oncogenic mutations by CRISPR base editing. By mutational scanning of IDH single amino acid variants in base-edited cells, we describe a repertoire of IDH second-site mutations responsible for therapy resistance through disabling uncompetitive enzyme inhibition. Recurrent mutations at NADPH binding sites within IDH heterodimers act in cis or trans to prevent the formation of stable enzyme-inhibitor complexes, restore R-2HG production in the presence of inhibitors, and drive therapy resistance in IDH-mutant AML cells and patients. We therefore uncover a new class of pathogenic mutations and mechanisms for acquired resistance to targeted cancer therapies. SIGNIFICANCE: Comprehensive scanning of IDH single amino acid variants in base-edited leukemia cells uncovers recurrent mutations conferring resistance to IDH inhibition through disabling NADPH-dependent uncompetitive inhibition. Together with targeted sequencing, structural, and functional studies, we identify a new class of pathogenic mutations and mechanisms for acquired resistance to IDH-targeting cancer therapies. This article is highlighted in the In This Issue feature, p. 1.

Immature Platelet Fraction as a Biomarker for Disease Severity in Pediatric Respiratory Coronavirus Disease 2019.

In this retrospective single-institution cohort study of 113 hospitalized pediatric patients with respiratory coronavirus disease 2019, those admitted to the intensive care unit or requiring mechanical ventilation had significantly higher immature platelet fractions than those who did not require intensive care unit-level care or ventilation. Immature platelet fraction may be an accessible biomarker for disease severity in pediatric respiratory coronavirus disease 2019.

Convergence of oncogenic cooperation at single-cell and single-gene levels drives leukemic transformation.

Cancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employ single-cell transcriptomic profiling to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid lineage-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid neoplasms with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically deregulate gene expression. We integrate results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We use this resource to relate developmental hierarchies to leukemia phenotypes, evaluate oncogenic cooperation at single-cell and single-gene levels, and identify GEM as a regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution at single-cell resolution in vivo.

SLC25A38 congenital sideroblastic anemia: Phenotypes and genotypes of 31 individuals from 24 families, including 11 novel mutations, and a review of the literature.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the presence of several alleles in different populations.

Silencing of LINE-1 retrotransposons is a selective dependency of myeloid leukemia.

Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.

Therapy Response and Outcome Explained by Leukemia Cell of Origin.

In this issue of Cancer Discovery, Cai and colleagues delineate a new mechanism that links cell of origin, the transcription factor EVI1, apoptotic priming, and therapeutic susceptibility in mixed lineage leukemia-rearranged acute myeloid leukemia. These findings establish a cell of origin-dependent program that may be leveraged by therapeutic combinations to overcome drug resistance in chemoresistant leukemias.See related article by Cai et al., p. 1500.

Diagnosis and treatment of pediatric myelodysplastic syndromes: A survey of the North American Pediatric Aplastic Anemia Consortium.

BACKGROUND: Myelodysplastic syndromes (MDS) represent a group of clonal hematopoietic stem cell disorders that commonly progress to acute myeloid leukemia (AML). The diagnostics, prognostics, and treatment of adult MDS are established but do not directly translate to children and adolescents. Pediatric MDS is a rare disease, characterized by unique cytogenetics and histology compared with adult MDS, and often arises secondary to germline predisposition or cytotoxic exposures. Our objective was to highlight aspects of diagnosis/management that would benefit from further systematic review toward the development of clinical practice guidelines for pediatric MDS. PROCEDURE: The North American Pediatric Aplastic Anemia Consortium (NAPAAC) is composed of collaborative institutions with a strong interest in pediatric bone marrow failure syndromes and hematologic malignancies. The NAPAAC MDS working group developed a national survey distributed to 35 NAPAAC institutions to assess data on (1) clinical presentation of pediatric MDS, (2) diagnostic evaluation, (3) criteria for diagnosis, (4) supportive care and treatment decisions, and (5) role of hematopoietic stem cell transplantation (HSCT). RESULTS: Twenty-eight of 35 institutions returned the survey. Most centers agreed on a common diagnostic workup, though there was considerable variation regarding the criteria for diagnosis. Although there was consensus on supportive care, treatment strategies, including the role of cytoreduction and HSCT, varied across centers surveyed. CONCLUSIONS: There is lack of national consensus on diagnosis and treatment of pediatric MDS. This survey identified key aspects of MDS management that will warrant systematic review toward the goal of developing national clinical practice guidelines for pediatric MDS.

Noncoding Variants Connect Enhancer Dysregulation with Nuclear Receptor Signaling in Hematopoietic Malignancies.

Mutations in protein-coding genes are well established as the basis for human cancer, yet how alterations within noncoding genome, a substantial fraction of which contain cis-regulatory elements (CRE), contribute to cancer pathophysiology remains elusive. Here, we developed an integrative approach to systematically identify and characterize noncoding regulatory variants with functional consequences in human hematopoietic malignancies. Combining targeted resequencing of hematopoietic lineage-associated CREs and mutation discovery, we uncovered 1,836 recurrently mutated CREs containing leukemia-associated noncoding variants. By enhanced CRISPR/dCas9-based CRE perturbation screening and functional analyses, we identified 218 variant-associated oncogenic or tumor-suppressive CREs in human leukemia. Noncoding variants at KRAS and PER2 enhancers reside in proximity to nuclear receptor (NR) binding regions and modulate transcriptional activities in response to NR signaling in leukemia cells. NR binding sites frequently colocalize with noncoding variants across cancer types. Hence, recurrent noncoding variants connect enhancer dysregulation with nuclear receptor signaling in hematopoietic malignancies. SIGNIFICANCE: We describe an integrative approach to identify noncoding variants in human leukemia, and reveal cohorts of variant-associated oncogenic and tumor-suppressive cis-regulatory elements including KRAS and PER2 enhancers. Our findings support a model in which noncoding regulatory variants connect enhancer dysregulation with nuclear receptor signaling to modulate gene programs in hematopoietic malignancies.See related commentary by van Galen, p. 646.This article is highlighted in the In This Issue feature, p. 627.

Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing.

Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe CRISPR/dCas9-based enhancer-targeting epigenetic editing systems, enCRISPRa and enCRISPRi, for efficient analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the improved systems display more robust perturbations of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Single or multi-loci perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoiesis. Hence, enhancer-targeting CRISPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.

Connecting the Dots From Fever of Unknown Origin to Myelodysplastic Syndrome: GATA2 Haploinsufficiency.

Leukemia-predisposing conditions, such as GATA2 haploinsufficiency, are known for their high penetrance and expressivity profiles. These disorders pose a difficult diagnostic challenge to even the most experienced clinician when they first present. We describe the case of a 17-year-old male presenting with features of nontuberculous mycobacterial infection, pulmonary fibrinoid granulomatous vasculitis, and myelodysplasia in the setting of a pathogenic GATA2 frameshift mutation confirmed by next-generation sequencing. The broad differential for GATA2 haploinsufficiency requires prompt recognition of key clinical features and laboratory abnormalities towards directing diagnosis and guiding appropriate and perhaps life-saving therapy.

Loss of EZH2 Reprograms BCAA Metabolism to Drive Leukemic Transformation.

Epigenetic gene regulation and metabolism are highly intertwined, yet little is known about whether altered epigenetics influence cellular metabolism during cancer progression. Here, we show that EZH2 and NRASG12D mutations cooperatively induce progression of myeloproliferative neoplasms to highly penetrant, transplantable, and lethal myeloid leukemias in mice. EZH1, an EZH2 homolog, is indispensable for EZH2-deficient leukemia-initiating cells and constitutes an epigenetic vulnerability. BCAT1, which catalyzes the reversible transamination of branched-chain amino acids (BCAA), is repressed by EZH2 in normal hematopoiesis and aberrantly activated in EZH2-deficient myeloid neoplasms in mice and humans. BCAT1 reactivation cooperates with NRASG12D to sustain intracellular BCAA pools, resulting in enhanced mTOR signaling in EZH2-deficient leukemia cells. Genetic and pharmacologic inhibition of BCAT1 selectively impairs EZH2-deficient leukemia-initiating cells and constitutes a metabolic vulnerability. Hence, epigenetic alterations rewire intracellular metabolism during leukemic transformation, causing epigenetic and metabolic vulnerabilities in cancer-initiating cells. SIGNIFICANCE: EZH2 inactivation and oncogenic NRAS cooperate to induce leukemic transformation of myeloproliferative neoplasms by activating BCAT1 to enhance BCAA metabolism and mTOR signaling. We uncover a mechanism by which epigenetic alterations rewire metabolism during cancer progression, causing epigenetic and metabolic liabilities in cancer-initiating cells that may be exploited as potential therapeutics.See related commentary by Li and Melnick, p. 1158.This article is highlighted in the In This Issue feature, p. 1143.

Abnormal Uterine Bleeding in Young Women with Blood Disorders.

Abnormal uterine bleeding is common in adolescents and is thought to affect 9% to 14% of women in their reproductive years. Certain unique aspects of underlying inherited or acquired blood disorders exacerbate the "expected" hormonal imbalance at this age, thereby increasing the morbidity of the underlying problem. A multifactorial etiology demands a collaborative approach between hematologists and gynecologists or adolescent medicine physicians to effectively manage abnormal uterine bleeding in young women with blood disorders.

In Situ Capture of Chromatin Interactions by Biotinylated dCas9.

Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human ß-globin genes establishes evidence for composition-based hierarchical organization. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally regulated super-enhancers reveals spatial features that causally control gene transcription. Thus, comprehensive and unbiased analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.

Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation.

Advances in genomic profiling present new challenges of explaining how changes in DNA and RNA are translated into proteins linking genotype to phenotype. Here we compare the genome-scale proteomic and transcriptomic changes in human primary haematopoietic stem/progenitor cells and erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Mitochondrial factors including TFAM and PHB2 are selectively regulated through protein translation during erythroid specification. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, and defective mitochondria and erythropoiesis. Mechanistically, mTORC1 signalling is enhanced to promote translation of mitochondria-associated transcripts through TOP-like motifs. Genetic and pharmacological perturbation of mitochondria or mTORC1 specifically impairs erythropoiesis in vitro and in vivo. Our studies support a mechanism for post-transcriptional control of erythroid mitochondria and may have direct relevance to haematologic defects associated with mitochondrial diseases and ageing.

Eradicating acute myeloid leukemia in a Mll(PTD/wt):Flt3(ITD/wt) murine model: a path to novel therapeutic approaches for human disease.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

Central nervous system imprinting of the G protein G(s)alpha and its role in metabolic regulation.

In Albright hereditary osteodystrophy, a monogenic obesity disorder linked to heterozygous mutations of G(s)alpha, the G protein that mediates receptor-stimulated cAMP generation, obesity develops only when the mutation is on the maternal allele. Likewise, mice with maternal (but not paternal) germline G(s)alpha mutation develop obesity, insulin resistance, and diabetes. These parent-of-origin effects are due to G(s)alpha imprinting, with preferential expression from the maternal allele in some tissues. As G(s)alpha is ubiquitously expressed, the tissue involved in this metabolic imprinting effect is unknown. Using brain-specific G(s)alpha knockout mice, we show that G(s)alpha imprinting within the central nervous system underlies these effects and that G(s)alpha is imprinted in the paraventricular nucleus of the hypothalamus. Maternal G(s)alpha mutation impaired melanocortin stimulation of energy expenditure but did not affect melanocortin's effect on food intake, suggesting that melanocortins may regulate energy balance in the central nervous system through both G(s)alpha-dependent and -independent pathways.