Single-Cell RNA-Seq Reveals AML Hierarchies Relevant to Disease Progression and Immunity.

Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies. VIDEO ABSTRACT.

Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis.

Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.

The origin and evolution of mutations in acute myeloid leukemia.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies.

Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b Importantly, primary human CBL mutated (CBLmut ) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies.

Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a.

Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16(INK4a) and p19(ARF), which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal.

Jmjd2/Kdm4 demethylases are required for expression of Il3ra and survival of acute myeloid leukemia cells.

Acute myeloid leukemias (AMLs) with a rearrangement of the mixed-linage leukemia (MLL) gene are aggressive hematopoietic malignancies. Here, we explored the feasibility of using the H3K9- and H3K36-specific demethylases Jmjd2/Kdm4 as putative drug targets in MLL-AF9 translocated leukemia. Using Jmjd2a, Jmjd2b, and Jmjd2c conditional triple-knockout mice, we show that Jmjd2/Kdm4 activities are required for MLL-AF9 translocated AML in vivo and in vitro. We demonstrate that expression of the interleukin 3 receptor α (Il3ra also known as Cd123) subunit is dependent on Jmjd2/Kdm4 through a mechanism involving removal of H3K9me3 from the promoter of the Il3ra gene. Importantly, ectopic expression of Il3ra in Jmjd2/Kdm4 knockout cells alleviates the requirement of Jmjd2/Kdm4 for the survival of AML cells, showing that Il3ra is a critical downstream target of Jmjd2/Kdm4 in leukemia. These results suggest that the JMJD2/KDM4 proteins are promising drug targets for the treatment of AML.

JMJD1C is required for the survival of acute myeloid leukemia by functioning as a coactivator for key transcription factors.

RUNX1-RUNX1T1 (formerly AML1-ETO), a transcription factor generated by the t(8;21) translocation in acute myeloid leukemia (AML), dictates a leukemic program by increasing self-renewal and inhibiting differentiation. Here we demonstrate that the histone demethylase JMJD1C functions as a coactivator for RUNX1-RUNX1T1 and is required for its transcriptional program. JMJD1C is directly recruited by RUNX1-RUNX1T1 to its target genes and regulates their expression by maintaining low H3K9 dimethyl (H3K9me2) levels. Analyses in JMJD1C knockout mice also establish a JMJD1C requirement for RUNX1-RUNX1T1's ability to increase proliferation. We also show a critical role for JMJD1C in the survival of multiple human AML cell lines, suggesting that it is required for leukemic programs in different AML cell types through its association with key transcription factors.

Constitutive BAK activation as a determinant of drug sensitivity in malignant lymphohematopoietic cells.

Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLXL•BAK complexes predicting navitoclax sensitivity, and extensive MCL1•BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK.

Acute myeloid leukemia presenting as horizontal diplopia.

Acute myeloid leukemia (AML) accounts for 16% of all leukemias in children. Prognosis in the pediatric population is better than that of older populations, with a younger age at diagnosis being a favorable prognostic factor [1]. Diplopia is a rare first presenting sign of AML. We present a 15 year old male complaining of diplopia and unilateral orbital swelling. Workup in the emergency department found normal neuroimaging but revealed a markedly elevated leukocytosis with anemia and thrombocytopenia. Peripheral smear showed increased blast cells >10%. This patient was ultimately diagnosed with AML. This case demonstrates an atypical presentation of AML and urges a thorough work up for patients presenting with unexplained diplopia.

Cancer-associated IDH2 mutants drive an acute myeloid leukemia that is susceptible to Brd4 inhibition.

Somatic mutations in the isocitrate dehydrogenase (IDH) genes IDH1 and IDH2 occur frequently in acute myeloid leukemia (AML) and other cancers. These genes encode neomorphic proteins that produce the presumed oncometabolite 2-hydroxyglutarate (2-HG). Despite the prospect of treating AML and other cancers by targeting IDH mutant proteins, it remains unclear how these mutants affect tumor development and maintenance in vivo, and no cancer models exist to study the action of IDH2 mutants in vivo. We show that IDH2 mutants can cooperate with oncogenic Flt3 or Nras alleles to drive leukemia in mice by impairing the differentiation of cells of the myeloid lineage. Pharmacologic or genetic inhibition of IDH2 triggers the differentiation and death of AML cells, albeit only with prolonged IDH2 inhibition. In contrast, inhibition of the bromodomain-containing protein Brd4 triggers rapid differentiation and death of IDH2 mutant AML. Our results establish a critical role for mutant IDH2 in leukemogenesis and tumor maintenance and identify an IDH-independent strategy to target these cancers therapeutically.

Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation.

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

Pediatric acute myeloid leukemia: updates on biology, risk stratification, and therapy.

PURPOSE OF REVIEW: Despite advances in therapy over the past decades, overall survival for children with acute myeloid leukemia (AML) has not exceeded 70%. In this review, we highlight recent insights into risk stratification for patients with pediatric AML and discuss data driving current and developing therapeutic approaches. RECENT FINDINGS: Advances in cytogenetics and molecular profiling, as well as improvements in detection of minimal residual disease after induction therapy, have informed risk stratification, which now relies heavily on these elements. The treatment of childhood AML continues to be based primarily on intensive, conventional chemotherapy. However, recent trials focus on limiting treatment-related toxicity through the identification of low-risk subsets who can safely receive fewer cycles of chemotherapy, allocation of hematopoietic stem-cell transplant to only high-risk patients and optimization of infectious and cardioprotective supportive care. SUMMARY: Further incorporation of genomic and molecular data in pediatric AML will allow for additional refinements in risk stratification to enable the tailoring of treatment intensity. These data will also dictate the incorporation of molecularly targeted therapeutics into frontline treatment in the hope of improving survival while decreasing treatment-related toxicity.

Acute Myeloid Leukaemia in Its Niche: the Bone Marrow Microenvironment in Acute Myeloid Leukaemia.

PURPOSE OF REVIEW: Acute myeloid leukaemia (AML) is a heterogeneous malignancy for which treatment options remain suboptimal. It is clear that a greater understanding of the biology of the AML niche will enable new therapeutic strategies to be developed in order to improve treatment outcomes for patients. RECENT FINDINGS: Recent evidence has highlighted the importance of the bone marrow microenvironment in protecting leukaemia cells, and in particular leukaemic stem cells from chemotherapy-induced cell death. This includes mesenchymal stem cells supporting growth and preventing apoptosis, and altered action and secretion profiles of other niche components including adipocytes, endothelial cells and T cells. Here, we provide a detailed overview of the current understanding of the AML bone marrow microenvironment. Clinical trials of agents that mobilise leukaemic stem cells from the bone marrow are currently ongoing and show early promise. Future challenges will involve combining these novel therapies targeted at the AML niche with conventional chemotherapy treatment.

Survival and Neurologic Recovery After Prompt Diagnosis and Aggressive Management of Severe Idiopathic Hyperammonemic Encephalopathy in a Patient with Acute Myeloid Leukemia.

A case of a 19-year-old female with low-risk acute myeloid leukemia is presented who was diagnosed with idiopathic hyperammonemic encephalopathy following the development of abrupt neurologic decline, respiratory alkalosis, and elevated plasma ammonia levels of unknown etiology. Delayed symptom recognition of this exceedingly rare condition contributes to the often fatal outcomes of idiopathic hyperammonemic encephalopathy. As illustrated by this case, prompt diagnosis and utilization of a variety of ammonia-modulating treatment modalities can result in remarkable clinical recovery. This case provides guidance to clinicians in counseling families about the possibility of neurologic recovery in similar clinical scenarios.

Clonal evolution of acute myeloid leukemia from diagnosis to relapse.

Based on the individual genetic profile, acute myeloid leukemia (AML) patients are classified into clinically meaningful molecular subtypes. However, the mutational profile within these groups is highly heterogeneous and multiple AML subclones may exist in a single patient in parallel. Distinct alterations of single cells may be key factors in providing the fitness to survive in this highly competitive environment. Although the majority of AML patients initially respond to induction chemotherapy and achieve a complete remission, most patients will eventually relapse. These points toward an evolutionary process transforming treatment-sensitive cells into treatment-resistant cells. As described by Charles Darwin, evolution by natural selection is the selection of individuals that are optimally adapted to their environment, based on the random acquisition of heritable changes. By changing their mutational profile, AML cell populations are able to adapt to the new environment defined by chemotherapy treatment, ultimately leading to cell survival and regrowth. In this review, we will summarize the current knowledge about clonal evolution in AML, describe different models of clonal evolution, and provide the methodological background that allows the detection of clonal evolution in individual AML patients. During the last years, numerous studies have focused on delineating the molecular patterns that are associated with AML relapse, each focusing on a particular genetic subgroup of AML. Finally, we will review the results of these studies in the light of Darwinian evolution and discuss open questions regarding the molecular background of relapse development.

Anthropometric factors and risk of myeloid leukaemias and myelodysplastic syndromes: a prospective study and meta-analysis.

There is insufficient evidence linking excess body weight to risk of myeloid malignancies. We investigated this association using data from the Cancer Prevention Study-II (CPS-II), and a meta-analysis of published cohort studies. Among 152 090 CPS-II participants, 387 acute myeloid leukaemias (AML), 100 chronic myeloid leukaemias (CML) and 170 MDS were identified over 21 years of follow-up. In CPS-II, body mass index (BMI) was weakly associated with risk of CML (hazard ratio [HR] = 1·04, 95% confidence interval [CI]: 0·99-1·09 per 1 unit increase in BMI), AML (HR = 1·01, 95% CI: 0·98-1·03) and MDS (HR = 1·03, 95% CI: 0·99-1·07). After controlling for other anthropometric factors, no clear association was observed for height, BMI at age 18 years or weight change. In the meta-analysis (n = 7117 myeloid leukaemias), BMI 25-29·9 kg/m2 (HRpooled  = 1·36, 95% CI: 1·12-1·59) and BMI ≥30 kg/m2 (HRpooled  = 1·43, 95% CI: 1·18-1·69) were associated with higher risk of myeloid leukaemia overall, compared to a BMI <25 kg/m2 . Likewise, BMI ≥25 kg/m2 was positively associated with both AML and CML risk individually in the meta-analysis. These results underscore the need for large studies to detect associations with rare cancers, and show a modest, but positive association between excess body weight and myeloid malignancy risk.

Cellular and Molecular State of Myeloid Leukemia Stem Cells.

Leukemia stem cells (LSCs) are leukemia-initiating population with the capacity to self-renew, differentiate, and stay quiescent. Human hematopoietic malignancies such as chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) are derived from this cell population. LSCs are also responsible for disease relapse due to its resistance to drug treatment. This rare cell population is phenotypically and functionally heterogeneous. Increasing evidence indicates that this heterogeneous cellular state of LSCs might determine the different drug sensitivity and is the major reason for disease relapse. In here, focusing on myeloid leukemia stem cells, we describe the biological features including cellular and molecular state, heterogeneity of LSCs, and the dynamic cross talk between LSCs and bone marrow microenvironment. These specific features of LSCs highlight the dynamic cellular state of LSCs, and further exploring on it might provide potential therapeutic targets that are important for eliminating LSCs.

RNA N 6-Methyladenosine Modification in Normal and Malignant Hematopoiesis.

As the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), N 6-methyladenosine (m6A) modification has been shown recently to posttranscriptionally regulate expression of thousands of messenger RNA (mRNA) transcripts in each mammalian cell type in a dynamic and reversible manner. This epigenetic mark is deposited by the m6A methyltransferase complex (i.e., the METTL3/METTL14/WTAP complex and other cofactor proteins) and erased by m6A demethylases such as FTO and ALKBH5. Specific recognition of these m6A-modified mRNAs by m6A-binding proteins (i.e., m6A readers) determines the fate of target mRNAs through affecting splicing, nuclear export, RNA stability, and/or translation. During the past few years, m6A modification has been demonstrated to play a critical role in many major normal bioprocesses including self-renewal and differentiation of embryonic stem cells and hematopoietic stem cells, tissue development, circadian rhythm, heat shock or DNA damage response, and sex determination. Thus, it is not surprising that dysregulation of the m6A machinery is also closely associated with pathogenesis and drug response of both solid tumors and hematologic malignancies. In this chapter, we summarize and discuss recent findings regarding the biological functions and underlying mechanisms of m6A modification and the associated machinery in normal hematopoiesis and the initiation, progression, and drug response of acute myeloid leukemia (AML), a major subtype of leukemia usually associated with unfavorable prognosis.

Epigenetic Abnormalities in Acute Myeloid Leukemia and Leukemia Stem Cells.

Recently advances in cancer genomics revealed the unexpected high frequencies of epigenetic abnormalities in human acute myeloid leukemia (AML). Accumulating data suggest that these leukemia-associated epigenetic factors play critical roles in both normal hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs). In turn, these abnormalities result in susceptibilities of LSC and related diseases to epigenetic inhibitors. In this chapter, we will focus on the mutations of epigenetic factors in AML, their functional roles and mechanisms in normal hematopoiesis and leukemia genesis, especially in LSC, and potential treatment opportunities specifically for AML with epigenetic dysregulations.

Leukemia Stem Cells in the Pathogenesis, Progression, and Treatment of Acute Myeloid Leukemia.

Despite the significant progress that has been made in understanding the biology of leukemia stem cells (LSCs), some key questions regarding the concept of LSCs have not as yet been satisfactorily addressed experimentally. As a result, the clinical relevance of LSCs remains less than clear due to controversies caused largely by technical limitations in efficiently identifying LSCs. This has impeded our ability to fully address the features of genetic heterogeneity and metabolic/epigenetic plasticity of pre-LSCs and LSCs. With the development and use of humanized immunocompromised mice, we are able to more precisely analyze LSCs for their functions and interaction with the bone marrow niche. In addition, some promising targets in LSCs have recently been identified, including Sonic Hedgehog (SHH) and BCL-2, which are highly expressed in AML cells. It is hopeful that new anti-LSC compounds will be tested fully in clinical trials for their efficacy in treating human leukemias.