CBFA2T3::GLIS2 pediatric acute megakaryoblastic leukemia is sensitive to BCL-XL inhibition by navitoclax and DT2216.

ABSTRACT: Acute megakaryoblastic leukemia (AMKL) is a rare, developmentally restricted, and highly lethal cancer of early childhood. The paucity and hypocellularity (due to myelofibrosis) of primary patient samples hamper the discovery of cell- and genotype-specific treatments. AMKL is driven by mutually exclusive chimeric fusion oncogenes in two-thirds of the cases, with CBFA2T3::GLIS2 (CG2) and NUP98 fusions (NUP98r) representing the highest-fatality subgroups. We established CD34+ cord blood-derived CG2 models (n = 6) that sustain serial transplantation and recapitulate human leukemia regarding immunophenotype, leukemia-initiating cell frequencies, comutational landscape, and gene expression signature, with distinct upregulation of the prosurvival factor B-cell lymphoma 2 (BCL2). Cell membrane proteomic analyses highlighted CG2 surface markers preferentially expressed on leukemic cells compared with CD34+ cells (eg, NCAM1 and CD151). AMKL differentiation block in the mega-erythroid progenitor space was confirmed by single-cell profiling. Although CG2 cells were rather resistant to BCL2 genetic knockdown or selective pharmacological inhibition with venetoclax, they were vulnerable to strategies that target the megakaryocytic prosurvival factor BCL-XL (BCL2L1), including in vitro and in vivo treatment with BCL2/BCL-XL/BCL-W inhibitor navitoclax and DT2216, a selective BCL-XL proteolysis-targeting chimera degrader developed to limit thrombocytopenia in patients. NUP98r AMKL were also sensitive to BCL-XL inhibition but not the NUP98r monocytic leukemia, pointing to a lineage-specific dependency. Navitoclax or DT2216 treatment in combination with low-dose cytarabine further reduced leukemic burden in mice. This work extends the cellular and molecular diversity set of human AMKL models and uncovers BCL-XL as a therapeutic vulnerability in CG2 and NUP98r AMKL.

Improved outcomes of UM171-expanded cord blood transplantation compared with other graft sources: real-world evidence.

Cord blood (CB) transplantation is hampered by low cell dose and high nonrelapse mortality (NRM). A phase 1-2 trial of UM171-expanded CB transplants demonstrated safety and favorable preliminary efficacy. The aim of the current analysis was to retrospectively compare results of the phase 1-2 trial with those after unmanipulated CB and matched-unrelated donor (MUD) transplants. Data from recipients of CB and MUD transplants were obtained from the Center for International Blood and Marrow Transplant Research (CIBMTR) database. Patients were directly matched for the number of previous allogeneic hematopoietic stem cell transplants (alloHCT), disease and refined Disease Risk Index. Patients were further matched by propensity score for age, comorbidity index, and performance status. Primary end points included NRM, progression-free survival (PFS), overall survival (OS), and graft-versus-host disease (GVHD)-free relapse-free survival (GRFS) at 1 and 2 years after alloHCT. Overall, 137 patients from CIBMTR (67 CB, 70 MUD) and 22 with UM171-expanded CB were included. NRM at 1 and 2 years was lower, PFS and GRFS at 2 years and OS at 1 year were improved for UM171-expanded CBs compared with CB controls. Compared with MUD controls, UM171 recipients had lower 1- and 2-year NRM, higher 2-year PFS, and higher 1- and 2-year GRFS. Furthermore, UM171-expanded CB recipients experienced less grades 3-4 acute GVHD and chronic GVHD compared with MUD graft recipients. Compared with real-world evidence with CB and MUD alloHCT, this study suggests that UM171-expanded CB recipients may benefit from lower NRM and higher GRFS. This trial was registered at www.clinicaltrials.gov as #NCT02668315.

Epigenetic changes in human model KMT2A leukemias highlight early events during leukemogenesis.

Chromosomal translocations involving KMT2A gene are one of the most common genetic alterations found in pediatric acute myeloid leukemias (AML) although the molecular mechanisms that initiate the disease remain incompletely defined. To elucidate these initiating events we have used a human model system of AML driven by the KMT2A-MLLT3 (KM3) fusion. More specifically, we investigated changes in DNA methylation, histone modifications, and chromatin accessibility at each stage of our model system and correlated these with expression changes. We observe the development of a profound hypomethylation phenotype in the early stages of leukemic transformation after KM3 addition along with loss of expression of stem cell associated genes along with skewed expression in other genes such as S100A8/9 implicated in leukemogenesis. In addition, early increases in the expression of the lysine demethylase KDM4B was functionally linked to these expression changes as well as other key transcription factors. Remarkably, our ATAC-seq data showed that there were relatively few leukemiaspecific changes and the vast majority corresponded to open chromatin regions and transcription factor clusters previously observed in other cell types. Integration of the gene expression and epigenetic changes revealed the adenylate cyclase gene ADCY9 as an essential gene in KM3-AML, and suggest the potential for autocrine signalling through the chemokine receptor CCR1 and CCL23 ligand. Together, our results suggest that KM3 induces subtle changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis.

Vesicular trafficking is a key determinant of the statin response in acute myeloid leukemia.

Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.

Harnessing Macrophages through the Blockage of CD47: Implications for Acute Myeloid Leukemia.

CD47 is a surface membrane protein expressed by all normal tissues. It is the so-called "don't eat me signal" because it protects the cells against phagocytosis. The CD47 interacts with the signal regulatory protein alpha (SIRPα) on the surface of macrophages, leading to downstream inhibitory signaling that dampens phagocytic capacity. Since macrophages exert immune surveillance against cancers, cancer cells overexpress CD47 to defend themselves against phagocytosis. Acute myeloid leukemia (AML) is a cancer of hematopoietic stem/progenitor cells (HSPC), and similar to other types of cancers, leukemic blasts show enhanced levels of CD47. In patients with AML, CD47 has been associated with a higher disease burden and poor overall survival. Blockage of CD47-SIRPα signaling leads to improved phagocytosis of AML cells and better overall survival in xenograft models. However, the introduction of a pro-phagocytic signal is needed to induce greater phagocytic capacity. These pro-phagocytic signals can be either Fc receptor stimulants (such as monoclonal antibodies) or natural pro-phagocytic molecules (such as calreticulin). Based on these pre-clinical findings, various clinical trials investigating the blockade of CD47-SIRPα interaction have been designed as monotherapy and in combination with other anti-leukemic agents. In this review, we will discuss CD47 biology, highlight its implications for AML pathophysiology, and explore the potential clinical translation of disrupting CD47-SIRPα to treat patients with AML.

VLA-4 Induces Chemoresistance of T Cell Acute Lymphoblastic Leukemia Cells via PYK2-Mediated Drug Efflux.

Cell adhesion plays a critical role in the development of chemoresistance, which is a major issue in anti-cancer therapies. In this study, we have examined the role of the VLA-4 integrin, a major adhesion molecule of the immune system, in the chemoresistance of T-ALL cells. We found that attachment of Jurkat and HSB-2 T-ALL cells to VCAM-1, a VLA-4 ligand, inhibits doxorubicin-induced apoptosis. However, their adhesion to fibronectin, which is mainly mediated via VLA-5, had no effect. Even the presence of the chemoattractant SDF1α (Stromal cell-derived factor-1α), which enhances the adhesion of T-ALL cells to fibronectin, did not modify the sensitivity of the cells attached on fibronectin towards doxorubicin-induced apoptosis. Mechanistically, we found that VLA-4 promoted T-ALL chemoresistance by inducing doxorubicin efflux. Our results showed that cell adhesion to both fibronectin and VCAM-1-induced Focal adhesion kinase (FAK) phosphorylation in T-ALL cells. However, only cell adhesion to VCAM-1 led to PYK2 phosphorylation. Inhibition studies indicated that FAK is not involved in doxorubicin efflux and chemoresistance, whereas PYK2 inhibition abrogated both VLA-4-induced doxorubicin efflux and chemoresistance. Together, these results indicate that the VLA-4/PYK2 pathway could participate in T-ALL chemoresistance and its targeting could be beneficial to limit/avoid chemoresistance and patient relapse.

Innate and adaptive immune responses toward nanomedicines.

Since the commercialization of the first liposomes used for drug delivery, Doxil/Caelyx® and Myocet®, tremendous progress has been made in understanding interactions between nanomedicines and biological systems. Fundamental work at the interface of engineering and medicine has allowed nanomedicines to deliver therapeutic small molecules and nucleic acids more efficiently. While nanomedicines are used in oncology for immunotherapy or to deliver combinations of cytotoxics, the clinical successes of gene silencing approaches like patisiran lipid complexes (Onpattro®) have paved the way for a variety of therapies beyond cancer. In parallel, the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the potential of mRNA vaccines to develop immunization strategies at unprecedented speed. To rationally design therapeutic and vaccines, chemists, materials scientists, and drug delivery experts need to better understand how nanotechnologies interact with the immune system. This review presents a comprehensive overview of the innate and adaptative immune systems and emphasizes the intricate mechanisms through which nanomedicines interact with these biological functions.

Role of the complement cascade in the biological fate of liposomes in rodents.

Nanomedicines, including liposomes, have been used to improve the clinical efficacy and safety of drugs. In some liposomal formulations, a hydrophilic polymer coating of poly(ethylene glycol) (PEG) is used to increase the circulation time. Understanding the biological mechanisms responsible for the clearance of PEGylated and non-PEGylated nanomedicines is necessary to develop better-performing materials. The purpose of this work is to explore the role of complement in the elimination of intravenously administered liposomes (PEGylated and non-PEGylated) in mice and rats. Here, the complement cascade was depleted by intraperitoneal injections of cobra venom factor (CVF) 12 and 24 hours before the intravenous injection of radiolabeled liposomes. In both mice and rats, non-PEGylated liposomes showed faster elimination than PEGylated liposomes. At a lipid dose of 20 mg kg-1, the abrogation of the complement cascade (in CVF group) did not alter the circulation time of either PEGylated or non-PEGylated liposomes. In contrast, at lower doses (2 mg kg-1), animals treated with CVF had slightly higher levels of circulating liposomes, especially during the 24 hours pharmacokinetic studies. The complement cascade seems to govern the uptake of non-PEGylated liposomes by splenic B cells. Altogether, these results suggest that although PEGylated and non-PEGylated liposomes can activate complement, the impact of this cascade on their circulation time is minor and mostly perceivable at later phases of distribution. This work enlightens biological pathways responsible for in vivo clearance of liposomes and will help in orienting future research in elucidating the nano-bio interface.

Hematopoietic stem cell transplantation using single UM171-expanded cord blood: a single-arm, phase 1-2 safety and feasibility study.

BACKGROUND: Benefits of cord blood transplantation include low rates of relapse and chronic graft-versus-host disease (GVHD). However, the use of cord blood is rapidly declining because of the high incidence of infections, severe acute GVHD, and transplant-related mortality. UM171, a haematopoietic stem cell self-renewal agonist, has been shown to expand cord blood stem cells and enhance multilineage blood cell reconstitution in mice. We aimed to investigate the safety and feasibility of single UM171-expanded cord blood transplantation in patients with haematological malignancies who do not have a suitable HLA-matched donor. METHODS: This single-arm, open-label, phase 1-2 safety and feasibility study was done at two hospitals in Canada. The study had two parts. In part 1, patients received two cord blood units (one expanded with UM171 and one unmanipulated cord blood) until UM171-expanded cord blood demonstrated engraftment. Once engraftment was documented we initiated part 2, reported here, in which patients received a single UM171-expanded cord blood unit with a dose de-escalation design to determine the minimal cord blood unit cell dose that achieved prompt engraftment. Eligible patients were aged 3-64 years, weighed 12 kg or more, had a haematological malignancy with an indication for allogeneic hematopoietic stem cell transplant and did not have a suitable HLA-matched donor, and a had a Karnofsky performance status score of 70% or more. Five clinical sites were planned to participate in the study; however, only two study sites opened, both of which only treated adult patients, thus no paediatric patients (aged <18 years) were recruited. Patients aged younger than 50 years without comorbidities received a myeloablative conditioning regimen (cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2, and 12 Gy total body irradiation) and patients aged older than 50 years and those with comorbidities received a less myeloablative conditioning regimen (cyclophosphamide 50 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, and 4 Gy total body irradiation). Patients were infused with the 7-day UM171-expanded CD34-positive cells and the lymphocyte-containing CD34-negative fraction. The primary endpoints were feasibility of UM171 expansion, safety of the transplant, kinetics of hematopoietic reconstitution (time to neutrophil and platelet engraftment) of UM171-expanded cord blood, and minimal pre-expansion cord blood unit cell dose that achieved prompt engraftment. We analysed feasibility in all enrolled patients and all other primary outcomes were analysed per protocol, in all patients who received single UM171-expanded cord blood transplantation. This trial has been completed and was registered with ClinicalTrials.gov, NCT02668315. FINDINGS: Between Feb 17, 2016, and Nov 11, 2018, we enrolled 27 patients, four of whom received two cord blood units for safety purposes in part 1 of the study. 23 patients were subsequently enrolled in part 2 to receive a single UM171-expanded cord blood transplant and 22 patients received a single UM171-expanded cord blood transplantation. At data cutoff (Dec 31, 2018), median follow-up was 18 months (IQR 12-22). The minimal cord blood unit cell dose at thaw that achieved prompt engraftment as a single cord transplant after UM171 expansion was 0·52 × 105 CD34-positive cells. We successfully expanded 26 (96%) of 27 cord blood units with UM171. Among the 22 patients who received single UM171-expanded cord blood transplantation, median time to engraftment of 100 neutrophils per µL was 9·5 days (IQR 8-12), median time to engraftment of 500 neutrophils per µL was 18 days (12·5-20·0), and no graft failure occurred. Median time to platelet recovery was 42 days (IQR 35-47). The most common non-haematological adverse events were grade 3 febrile neutropenia (16 [73%] of 22 patients) and bacteraemia (nine [41%]). No unexpected adverse events were observed. One (5%) of 22 patients died due to treatment-related diffuse alveolar haemorrhage. INTERPRETATION: Our preliminary findings suggest that UM171 cord blood stem cell expansion is feasible, safe, and allows for the use of small single cords without compromising engraftment. UM171-expanded cord blood might have the potential to overcome the disadvantages of other cord blood transplants while maintaining the benefits of low risk of chronic GVHD and relapse, and warrants further investigation in randomised trials. FUNDING: Canadian Institutes of Health Research, Canadian Cancer Society and Stem Cell Network.

Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

Chemoresistance is a major hurdle in anti-cancer therapy. Growing evidence indicates that integrin-mediated cell adhesion to extracellular matrix plays a major role in chemoresistance. However, the underlying mechanisms are not fully understood. We have previously shown that the collagen-binding integrin α2ß1 promoted doxorubicin resistance in acute T cell lymphoblastic leukemia (T-ALL). In this study, we found that acute myeloid leukemia (AML) cell lines also express α2ß1 integrin and collagen promoted their chemoresistance as well. Furthermore, we found that high levels of α2 integrin correlate with worse overall survival in AML. Our results showed that doxorubicin-induced apoptosis in leukemic cells is associated with activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and that collagen inhibited this pathway. The protective effect of collagen is associated with the inhibition of Rac1-induced DNA damage as evaluated by the comet assay and the phosphorylated levels of histone H2AX (γ-H2AX). Together these results show that by inhibiting pro-apoptotic Rac1, α2ß1 integrin can be a major pathway protecting leukemic cells from genotoxic agents and may thus represent an important therapeutic target in anti-cancer treatment.

Pediatric leukemia: Moving toward more accurate models.

Leukemia is a complex genetic disease caused by errors in differentiation, growth, and apoptosis of hematopoietic cells in either lymphoid or myeloid lineages. Large-scale genomic characterization of thousands of leukemia patients has produced a tremendous amount of data that have enabled a better understanding of the differences between adult and pediatric patients. For instance, although phenotypically similar, pediatric and adult myeloid leukemia patients differ in their mutational profiles, typically involving either chromosomal translocations or recurrent single-base-pair mutations, respectively. To elucidate the molecular mechanisms underlying the biology of this cancer, continual efforts have been made to develop more contextually and biologically relevant experimental models. Leukemic cell lines, for example, provide an inexpensive and tractable model but often fail to recapitulate critical aspects of tumor biology. Likewise, murine leukemia models of leukemia have been highly informative but also do not entirely reproduce the human disease. More recent advances in the development of patient-derived xenografts (PDXs) or human models of leukemias are poised to provide a more comprehensive, and biologically relevant, approach to directly assess the impact of the in vivo environment on human samples. In this review, the advantages and limitations of the various current models used to functionally define the genetic requirements of leukemogenesis are discussed.

Beta1 integrin blockade overcomes doxorubicin resistance in human T-cell acute lymphoblastic leukemia.

Growing evidence indicates that cell adhesion to extracellular matrix (ECM) plays an important role in cancer chemoresistance. Leukemic T cells express several adhesion receptors of the ß1 integrin subfamily with which they interact with ECM. However, the role of ß1 integrins in chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL) is still ill defined. In this study, we demonstrate that interactions of human T-ALL cell lines and primary blasts with three-dimensional matrices including Matrigel and collagen type I gel promote their resistance to doxorubicin via ß1 integrin. The blockade of ß1 integrin with a specific neutralizing antibody sensitized xenografted CEM leukemic cells to doxorubicin, diminished the leukemic burden in the bone marrow and resulted in the extension of animal survival. Mechanistically, Matrigel/ß1 integrin interaction enhanced T-ALL chemoresistance by promoting doxorubicin efflux through the activation of the ABCC1 drug transporter. Finally, our findings showed that Matrigel/ß1 interaction enhanced doxorubicin efflux and chemoresistance by activating the FAK-related proline-rich tyrosine kinase 2 (PYK2) as both PYK2 inhibitor and siRNA diminished the effect of Matrigel. Collectively, these results support the role of ß1 integrin in T-ALL chemoresistance and suggest that the ß1 integrin pathway can constitute a therapeutic target to avoid chemoresistance and relapsed-disease in human T-ALL.

Hepatic leukemia factor is a novel leukemic stem cell regulator in DNMT3A, NPM1, and FLT3-ITD triple-mutated AML.

FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56 highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.

Identification of novel biomarkers for MLL-translocated acute myeloid leukemia.

Acute myeloid leukemias (AMLs) with translocations of the mixed lineage leukemia (MLL/KMT2A) gene are common in young patients and are generally associated with poor clinical outcomes. The molecular biology of MLL fusion genes remains incompletely characterized and is complicated by the fact that more than 100 different partner genes have been identified in fusions with MLL. The continuously growing list of MLL fusions also represents a clinical challenge with respect to identification of novel fusions and tracking of the fusions to monitor progression of the disease after treatment. Recently, we have developed a novel single-donor model leukemia system that permits the development of human AML from normal cord blood cells. Gene expression analysis of this model and of MLL-AML patient samples has identified a number of candidate biomarker genes with highly biased expression on leukemic cells. Here, we present data demonstrating the potential clinical utility of several of these candidate genes for identifying known and novel MLL fusions.

S100A9 induces differentiation of acute myeloid leukemia cells through TLR4.

S100A8 and S100A9 are calcium-binding proteins predominantly expressed by neutrophils and monocytes and play key roles in both normal and pathological inflammation. Recently, both proteins were found to promote tumor progression through the establishment of premetastatic niches and inhibit antitumor immune responses. Although S100A8 and S100A9 have been studied in solid cancers, their functions in hematological malignancies remain poorly understood. However, S100A8 and S100A9 are highly expressed in acute myeloid leukemia (AML), and S100A8 expression has been linked to poor prognosis in AML. We identified a small subpopulation of cells expressing S100A8 and S100A9 in AML mouse models and primary human AML samples. In vitro and in vivo analyses revealed that S100A9 induces AML cell differentiation, whereas S100A8 prevents differentiation induced by S100A9 activity and maintains AML immature phenotype. Treatment with recombinant S100A9 proteins increased AML cell maturation, induced growth arrest, and prolonged survival in an AML mouse model. Interestingly, anti-S100A8 antibody treatment had effects similar to those of S100A9 therapy in vivo, suggesting that high ratios of S100A9 over S100A8 are required to induce differentiation. Our in vitro studies on the mechanisms/pathways involved in leukemic cell differentiation revealed that binding of S100A9 to Toll-like receptor 4 (TLR4) promotes activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2, and Jun N-terminal kinase signaling pathways, leading to myelomonocytic and monocytic AML cell differentiation. These findings indicate that S100A8 and S100A9 are regulators of myeloid differentiation in leukemia and have therapeutic potential in myelomonocytic and monocytic AMLs.

Evaluation of human epidermal growth factor receptor 2 (HER2) single nucleotide polymorphisms (SNPs) in normal and breast tumor tissues and their link with breast cancer prognostic factors.

Amplification of the human epidermal growth factor receptor 2 (HER2) gene is associated with worse prognosis and decreased overall survival in breast cancer patients. The HER2 gene contains several polymorphisms; two of the best-characterized HER2 polymorphisms are Ile655Val and Ala1170Pro. The aim of this study was to evaluate the association between these two HER2 polymorphisms in normal breast and breast cancer tissues and known breast cancer prognostic factors in a retrospective cohort study of 73 women with non-metastatic HER2-positive breast cancer. HER2 polymorphisms were assessed in breast cancer tissue and normal breast tissue using TaqMan assay. Ala1170Pro polymorphism in normal breast tissue was associated with age at diagnosis (p = 0.007), tumor size (p = 0.004) and lymphovascular invasion (p = 0.06). Similar significant associations in cancer tissues were observed. No association between the Ile655Val polymorphism and prognostic factors were observed. However, we found significant differences in the distribution of Ile655Val (p = 0.03) and Ala1170Pro (p = 0.01) genotypes between normal breast and breast tumor tissues. This study demonstrates that only the Ala1170Pro polymorphism is associated with prognostic factors in HER2-positive breast cancer patients. Moreover, our results suggest that both HER2 polymorphisms could play a significant role in carcinogenesis in non-metastatic HER2-positive breast cancer women.

GPR56 identifies primary human acute myeloid leukemia cells with high repopulating potential in vivo.

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal. Here, we combined next-generation sequencing technology with in vivo assessment of LSC frequencies and identified the adhesion G protein-coupled receptor 56 (GPR56) as a novel and stable marker for human LSCs for the majority of AML samples. High GPR56 expression was significantly associated with high-risk genetic subgroups and poor outcome. Analysis of GPR56 in combination with CD34 expression revealed engraftment potential of GPR56(+)cells in both the CD34(-)and CD34(+)fractions, thus defining a novel LSC compartment independent of the CD34(+)CD38(-)LSC phenotype.

The transcriptomic landscape and directed chemical interrogation of MLL-rearranged acute myeloid leukemias.

Using next-generation sequencing of primary acute myeloid leukemia (AML) specimens, we identified to our knowledge the first unifying genetic network common to the two subgroups of KMT2A (MLL)-rearranged leukemia, namely having MLL fusions or partial tandem duplications. Within this network, we experimentally confirmed upregulation of the gene with the most subtype-specific increase in expression, LOC100289656, and identified cryptic MLL fusions, including a new MLL-ENAH fusion. We also identified a subset of MLL fusion specimens carrying mutations in SPI1 accompanied by inactivation of its transcriptional network, as well as frequent RAS pathway mutations, which sensitized the leukemias to synthetic lethal interactions between MEK and receptor tyrosine kinase inhibitors. This transcriptomics-based characterization and chemical interrogation of human MLL-rearranged AML was a valuable approach for identifying complementary features that define this disease.