Form Of Acute Myeloid Leukemia Research Articles

Proteomic Profiling Reveals Protein Expression Patterns Correlated with Outcome in Acute Promyelocytic Leukemia (APL)

Background: Acute promyelocytic leukemia (APL) is the most favorable prognostic form of acute myeloid leukemia (AML). However, 5-20% of the patients relapse, which indicates that the clinical need to identify those who are likely to relapse remains. In this study we aimed to define protein expression patterns of APL with the goal to find certain protein patterns that would be prognostic for outcome.Methods: Custom reverse phase protein arrays (RPPA) were performed on 20 newly diagnosed APL patients, 205 newly diagnosed AML patients, and 10 normal CD34+ bone marrow samples, probed with 230 strictly validated antibodies. First, proteins were divided into 31 functionally related groups to analyze them in the context of other proteins. Progeny clustering analysis was applied to each of the functional groups to identify an optimal number of protein clusters; cases with similar expression of proteins. Then, block clustering was done on a binary matrix that indicated the protein cluster membership for all protein groups, for selected APL patients only, to assess whether we could identify subsets of APL cases (signatures) that expressed similar patterns of correlated protein clusters from various protein groups. Survival curves were generated for the identified signatures and individual proteins were compared between signatures using the student t-test with the Bonferroni correction (P<0.05).Results: Protein expression levels were measured for APL and AML patients relative to the normal CD34+ samples. Progeny clustering analysis revealed protein clusters for each functionally related protein group. All protein clusters that included APL patients were also found in AML patients. Only for the functional protein groups “Apoptosis regulating”, “HIPPO” and “TP53”, we observed protein patterns specific to APL. Block-clustering on APL patients showed strong correlations between various protein clusters and identified two subsets of patients, which we called protein signatures. Interestingly, survival curves showed that all of the relapse patients (N=4/20, 80.0%) were present in signature 2 (N=4/13, 30.8% vs. signature 1 N=0/7, 0%). Due to the small number of patients in our cohort, no level of significance was reached (P=0.084). Moreover, the number of promyelocytes in the bone marrow was lower in signature 1 (median 47 vs. 71 overall, P=0.070) and fibrinogen levels were higher in signature 1 (median 235 vs. 159, P=0.052) compared to signature 2. Direct comparison of individual proteins between signature 1 and 2 found 38 differentially expressed proteins. Notably, most protein had opposite protein levels relative to the normal cells in both signatures. For instance, ERG, STMN1, SSBP2, PRKAA1_2.pT172 and hnRNPK were higher in signature 2 and lower in signature 1, whereas CTSG, ITGB3 and ITGA2 were higher in signature 1.Conclusion: We demonstrated that although APL is genetically and therapeutically unique form of AML, most expression patterns were comparable between APL and AML. Most interestingly, we found that a subset of APL patients could be distinguished as high-risk solely based on their protein expression patterns. By knowing the signature membership at an early stage could, we could then decide whether additional therapy is required or not. Recognition of proteins that are higher or lower expressed in both signatures could be used to identify novel therapeutic targets. However, validation of our findings in a larger cohort is required. DisclosuresNo relevant conflicts of interest to declare.

CG '806, a First-in-Class Non-Covalent Pan-FLT3/BTK Inhibitor, Exerts Superior Potency Against B-Cell Malignant Cells

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/Bruton's tyrosine kinase (BTK) multi-kinase inhibitor that potently inhibits wild type (WT) and mutant forms of FLT3 and BTK. As described previously (2017 AACR Hematologic Malignancies abstract #25 and #44), CG'806 inhibits on FLT3-ITD-driven acute myeloid leukemia (AML) and other forms of AML. Because CG'806 targets the WT BTK (IC50 = 8 nM) and the C481S-BTK (IC50 = 2.5 nM), which is a mutant form resistant to ibrutinib and other covalent BTK inhibitors, this small molecule was profiled for its actions on various types of B-cell malignancies in which B cell receptor (BCR) and BTK signaling are reported to play pivotal pathogenic roles. Against 10 malignant B-cell lines, while CG'806 reduced the level of BTK-Y223 phosphorylation, it was less potent than ibrutinib. In contrast, CG'806 was 50-6,000 times more potent (lower IC50) in the ability to kill the malignant B cells across all tested cell lines evaluated by MTS-based assays of cell proliferation and inhibition of colony formation. Hence, the efficacy of killing malignant B cells by CG'806 and by ibrutinib did not correlate with the degree of BTK inhibition. Pathway analyses (KinomeScan and immunoblotting) demonstrated that CG'806, in addition to inhibiting intracellular BTK, potently inhibited the activity of aurora kinases A, B and C, phospho-H3S10, FLT3 and ERK that are differentially expressed and operative in various cell lines representing mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), B-acute lymphocytic leukemia (B-ALL) and Burkitt's lymphoma. Indeed, measuring BrdU incorporation and Annexin V by flow cytometry, CG'806 directly induced polyploidy and triggered apoptosis in malignant B cells at picomolar (pM) to low nanomolar (nM) concentrations, while ibrutinib potently inhibited BTK activity but was unable to induce apoptosis at lower than micromolar (µM) concentrations. These data distinguish CG'806 from ibrutinib and other covalent and non-covalent BTK inhibitors, and illustrate that CG'806 has activity across a wide range of malignant B cell lines including those intolerant, refractory or resistant (due to the presence of a BTK-C481S mutation) to ibrutinib and other BTK inhibitors. [Display omitted] DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Combinatorial Use of Bexarotene Plus ATRA for the Treatment of Acute Myeloid Leukemia

Aim: All-trans retinoic acid (ATRA) results in high response rates in acute promyelocytic leukemia (APL), but has not had a significant impact on non-APL forms of acute myeloid leukemia (AML), even though retinoic acid receptors have been implicated in hematopoietic maturation and self-renewal. RXRs bind as heterodimers with RARs, and are expressed at higher levels in AML cells than are RARs. We sought to determine whether natural RXR ligands exist in primary leukemia cells, whether RXR ligands might augment or inhibit leukemic cell growth, and whether RXRs might be rational targets for AML therapeutics.Background: Retinoid receptors are nuclear hormone receptors, induced during terminal myeloid maturation with highest expressed in M4 and M5 AML. Retinoids serve as intracellular messengers and are activating ligands for the retinoic acid receptors (RAR) and retinoid X receptors (RXR).Methods: We developed an in vivo reporter assay that measures intracellular, activating nuclear receptor ligands in mouse bone marrow cells. We generated transgenic mice with GFP regulated by a UAS promoter. The yeast Gal4 transcription factor recognizes UAS promoter sequences. When a fusion of the Gal4-DNA binding domain (DBD) and a retinoid receptor ligand-binding domain (LBD) is expressed in UAS-GFP bone marrow cells, and an activating ligand is present, the cells express GFP. Leukemia was generated in bone marrow cells from these mice and from Mx1-Cre x Rxrαfl/fl x Rxrβfl/fl mice using MSCV-MLL-AF9 retrovirus.Results: We found that intermediate levels of RXRA ligands are present in mouse MLL-AF9 leukemia cells in vivo. To determine if activation of RXRA is required for MLL-AF9 cell growth, we transduced MSCV-MLL-AF9 retrovirus into bone marrow cells from Mx1-Cre+ x Rxrαfl/fl x Rxrβfl/fl mice and transplanted these into recipient mice. Mx1-Cre is “leaky.” Without external activation of Mx1-Cre (i.e. without pIpC treatment) we found that all subsequent leukemias were associated with deleted (“floxed out”) Rxra and Rxrb alleles, suggesting that the deleted allele was positively selected for. In secondary transplants, we found that MLL-AF9 leukemias with deleted Rxrα and Rxrβ were associated with shorter survival when compared with MLL-AF9 leukemias with wild type Rxrα and Rxrβ, suggesting that naturally occurring RXR ligands suppressed leukemic growth. To determine if pharmacologic activation of retinoid receptors could inhibit MLL-AF9 cell growth, we treated MLL-AF9 cells ex vivo with ATRA and bexarotene. We found that ATRA modestly inhibited growth, and this effect reached a plateau at 62 nM (this may partially explain the limited clinical efficacy of ATRA in non-APL AML) (see Figure). In contrast, the EC50 of bexarotene was 125 nM and the EC50 decreased to 54 nM when bexarotene was combined with 62 nM ATRA. Higher doses of ATRA decreased the bexarotene EC50 further to 43 nM and resulted in near complete culture collapse. Finally, to determine whether combination bexarotene and ATRA inhibited cell growth by inducing differentiation or apoptosis, we examined cytomorphology, cell division by Celltrace Violet staining (similar to CFSE), and Annexin V. Unexpectedly, we observed no evidence of differentiation, but rather increased Annexin V associated with ATRA and bexarotene-induced growth arrest.Conclusion: RXRs act as tumor suppressors in MLL-AF9 murine leukemia cells in vivo, and the activation of RXR/RAR heterodimers with bexarotene and all-trans retinoic acid leads to synergistic apoptosis and growth arrest. Additional experiments are now being performed to test the efficacy of this dug combination in vivo and assess their potential use in elderly relapsed/refractory AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tumor suppressor function of Gata2 in acute promyelocytic leukemia

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Erythroleukemia: an Update

Acute erythroleukemia (AEL) is a rare form of acute myeloid leukemia recognized by erythroblastic proliferation. Many controversies remain around diagnosis influencing prognostic and therapeutic implications relating to this unique leukemia subset. The 2016 WHO classification includes more clear and restrictive diagnostic criteria for AEL. Primary acute erythroid leukemia is associated with complex and high-risk karyotypes including chromosomes 5q and 7q abnormalities. Mutational data shows that AEL is characterized by far lower NPM1 and FLT3-ITD mutation rates and higher mutational rates in TP53 compared with other AML subtypes. Hypomethylating agents have shown therapeutic value in AEL. In this article, we discuss the evolving diagnostic concepts of erythroleukemia, genomics, clinical outcome, and promising therapeutic targets through an appraisal of the current literature.

Recent advances in prognostication and treatment of polycythemia vera.

Polycythemia vera (PV) is a BCR-ABL–negative myeloproliferative neoplasm marked by acquisition of an activating mutation of JAK2, which leads to not only erythrocytosis but also frequently to leukocytosis and thrombocytosis, and is associated with a high symptom burden and increased thrombotic risk. PV has the potential to progress to myelofibrosis or an aggressive form of acute myeloid leukemia. Mutational profiling of patients with PV has led to the development of risk stratification tools to determine an individual’s risk of developing progressive disease. Although the current goals of PV treatment are to alleviate symptoms and reduce thrombotic risk, there are growing efforts to identify disease-modifying agents which will also prevent progression of disease. Here, we give an overview of the developing prognostic tools and therapeutic landscape for PV, focusing on four drug classes: pegylated interferon-alpha 2, MDM2 antagonists, hepcidin mimetics, and histone deacetylase inhibitors.

LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms.

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. SIGNIFICANCE: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.This article is highlighted in the In This Issue feature, p. 1307.

Endogenous and combination retinoids are active in myelomonocytic leukemias

Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL) but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The presence of natural retinoids in vivo could influence the efficacy of pharmacologic agonists and antagonists. We found that natural RXRA ligands, but not RARA ligands, were present in murine MLL-AF9-derived myelomonocytic leukemias in vivo and that the concurrent presence of receptors and ligands acted as tumor suppressors. Pharmacologic retinoid responses could be optimized by concurrent targeting of RXR ligands (e.g., bexarotene) and RARA ligands (e.g., all-trans retinoic acid), which induced either leukemic maturation or apoptosis depending on cell culture conditions. Co-repressor release from the RARA:RXRA heterodimer occurred with RARA activation, but not RXRA activation, providing an explanation for the combination synergy. Combination synergy could be replicated in additional, but not all, AML cell lines and primary samples, and was associated with improved survival in vivo, although tolerability of bexarotene administration in mice remained an issue. These data provide insight into the basal presence of natural retinoids in leukemias in vivo and a potential strategy for clinical retinoid combination regimens in leukemias beyond APL.

Loss of LKB1/STK11 Facilitates Leukemic Progression of the Myeloproliferative Neoplasms

Loss of LKB1/STK11 Facilitates Leukemic Progression of the Myeloproliferative Neoplasms

Investigators aim to increase CAR T-cell research and extend its reach: A new initiative by the National Cancer Institute could help this life-saving treatment reach more patients with advanced cancers.

Investigators aim to increase CAR T-cell research and extend its reach: A new initiative by the National Cancer Institute could help this life-saving treatment reach more patients with advanced cancers.

Buffering noise: KAT2A modular contributions to stabilization of transcription and cell identity in cancer and development

KAT2A is a histone acetyltransferase recently identified as a vulnerability in at least some forms of Acute Myeloid Leukemia (AML). Its loss or inhibition prompts leukemia stem cells out of self-renewal and into differentiation with ultimate exhaustion of the leukemia pool. We have recently linked the Kat2a requirement in AML to control of transcriptional noise, reflecting an evolutionary-conserved role of Kat2a in promoting burst-like promoter activity and stabilizing gene expression. We suggest that through this role, Kat2a contributes to preservation of cell identity. KAT2A exerts its acetyltransferase activity in the context of two macromolecular complexes, Spt-Ada-Gcn5-Acetyltransferase (SAGA) and Ada-Two-A-Containing (ATAC), but the specific contribution of each complex to stabilization of gene expression is currently unknown. By reviewing specific gene targets and requirements of the two complexes in cancer and development, we suggest that SAGA regulates lineage-specific programs, and ATAC maintains biosynthetic activity through control of ribosomal protein and translation-associated genes, on which cells may be differentially dependent. While our data suggest that KAT2A-mediated regulation of transcriptional noise in AML may be exerted through ATAC, we discuss potential caveats and probe general vs. complex-specific contributions of KAT2A to transcriptional stability, with implications for control and perturbation of cell identity.

3033 – SINGLE-CELL MULTI-OMIC ANALYSIS UNRAVELS GENETIC AND MOLECULAR HETEROGENEITY IN TRANSFORMED MYELOPROLIFERATIVE NEOPLASMS

One out of five patients with myeloproliferative neoplasms (MPN) will progress to an aggressive and largely untreatable form of Acute Myeloid Leukaemia (post-MPN secondary AML, or sAML). Transformation is associated with acquisition of TP53 missense mutations in approximately 50% of cases, and markedly increased genetic and molecular heterogeneity. This makes it challenging to effectively eliminate all subpopulations of tumour cells, and likely underlies the mostly universal treatment failure in sAML. Characterizing the complex genetic and cellular hierarchies associated with TP53 transformation is crucial for the development of effective targeted therapies. To resolve these multiple layers of intratumoral heterogeneity, we recently developed TARGET-seq (Rodriguez-Meira et al, 2019), a single-cell multi-omic method that correlates genetic, transcriptional and cell-surface proteomic readouts from the same single cell with extremely high resolution. TARGET-seq analysis of over 7000 Lineage-CD34+ cells (indexed for CD38/90/45RA/123) from TP53-mutant sAML patients revealed striking patterns of clonal evolution in different immunophenotypically defined stem/progenitor cell types, and identified preleukemic and leukemic subclones with markedly distinct molecular signatures. Acquisition of TP53 mutations was invariably associated with highly complex clonal evolution, enrichment of cell-cycle associated transcription and downregulation of key stem cell regulators. Interestingly, preleukemic subclones from patients undergoing transformation were highly enriched in WNT-beta catenin signaling and depleted in DNA-repair genes, as compared to non-transformed MPN patients. This suggests that preleukemic clones could be primed for disease transformation, and could potentially be targeted to prevent disease progression. Ultimately, single-cell multi-omic analysis identified key molecular regulators of TP53 mediated transformation, providing unique insights into the evolution of these tumors and how they might be therapeutically targeted.

Mechanistic insights into chromatin targeting by leukemic NUP98-PHF23 fusion

Chromosomal NUP98-PHF23 translocation is associated with an aggressive form of acute myeloid leukemia (AML) and poor survival rate. Here, we report the molecular mechanisms by which NUP98-PHF23 recognizes the histone mark H3K4me3 and is inhibited by small molecule compounds, including disulfiram that directly targets the PHD finger of PHF23 (PHF23PHD). Our data support a critical role for the PHD fingers of NUP98-PHF23, and related NUP98-KDM5A and NUP98-BPTF fusions in driving leukemogenesis, and demonstrate that blocking this interaction in NUP98-PHF23 expressing AML cells leads to cell death through necrotic and late apoptosis pathways. An overlap of NUP98-KDM5A oncoprotein binding sites and H3K4me3-positive loci at the Hoxa/b gene clusters and Meis1 in ChIP-seq, together with NMR analysis of the H3K4me3-binding sites of the PHD fingers from PHF23, KDM5A and BPTF, suggests a common PHD finger-dependent mechanism that promotes leukemogenesis by this type of NUP98 fusions. Our findings highlight the direct correlation between the abilities of NUP98-PHD finger fusion chimeras to associate with H3K4me3-enriched chromatin and leukemic transformation.

ATPR induces acute promyelocytic leukemia cells differentiation and growth arrest by blockade of SHP2/Rho/ROCK1 pathway

Acute promyelocytic leukemia (APL) is a form of acute myeloid leukemia with a unique chromosome translocation t (15;17), commonly complicated by a complex coagulopathy. 4-Amino-2-trifuoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, was synthesized by our group and known to possess obvious biological anti-tumor activities. It has previously been shown that ATPR could induce differentiation and inhibit proliferation of APL cells, although the mechanism responsible for this effect was not well understood. In this study, we demonstrated that ATPR remarkably inhibited the expression and activity of SHP2. Further experiments showed silencing SHP2 or using SHP2 inhibition (SHP099) enhanced the effect of ATPR on cell proliferation and maturation. In addition, we also demonstrated that Rho/ROCK1 might be regulated by SHP2. Using Y-27632, a ROCK inhibitor, further proved that ROCK1 played an important role in ATPR-induced differentiation and proliferation suppression. In conclusion, the results from this study revealed that ATPR induced APL cells terminal differentiation and growth arrest by blockade of SHP2/Rho/ ROCK1 pathway.

Acute Promyelocytic Leukemia: A Constellation of Molecular Events around a Single PML-RARA Fusion Gene

Although acute promyelocytic leukemia (APL) is one of the most characterized forms of acute myeloid leukemia (AML), the molecular mechanisms involved in the development and progression of this disease are still a matter of study. APL is defined by the PML-RARA rearrangement as a consequence of the translocation t(15;17)(q24;q21). However, this abnormality alone is not able to trigger the whole leukemic phenotype and secondary cooperating events might contribute to APL pathogenesis. Additional somatic mutations are known to occur recurrently in several genes, such as FLT3, WT1, NRAS and KRAS, whereas mutations in other common AML genes are rarely detected, resulting in a different molecular profile compared to other AML subtypes. How this mutational spectrum, including point mutations in the PML-RARA fusion gene, could contribute to the 10%–15% of relapsed or resistant APL patients is still unknown. Moreover, due to the uncertain impact of additional mutations on prognosis, the identification of the APL-specific genetic lesion is still the only method recommended in the routine evaluation/screening at diagnosis and for minimal residual disease (MRD) assessment. However, the gene expression profile of genes, such as ID1, BAALC, ERG, and KMT2E, once combined with the molecular events, might improve future prognostic models, allowing us to predict clinical outcomes and to categorize APL patients in different risk subsets, as recently reported. In this review, we will focus on the molecular characterization of APL patients at diagnosis, relapse and resistance, in both children and adults. We will also describe different standardized molecular approaches to study MRD, including those recently developed. Finally, we will discuss how novel molecular findings can improve the management of this disease.

FEATURES OF A DIFFERENTIAL DIAGNOSIS OF PEDIATRIC ACUTE MONOCYTIC LEUKEMIA (AML-M5a) ON THE EXAMPLE OF A CLINICAL CASE

The aim of this work was to assess the significance of investigating clinical and laboratory parameters for diagnosing acute monocytic leukemia in children on the basis of a clinical case. The article demonstrates specific features of differentiating AML M5a from other forms of acute myeloid leukemia. According to the results of hematological, morphological and cytofluorimetric studies of blood and bone marrow samples, the diagnosis of acute myeloid leukemia was established. The morphological and phenotypic characteristics of blast cells hampered the diagnosis of an AML form. However, a comprehensive analysis of the expressed CD antigens allowed acute monocytic leukemia to be identified, which diagnosis was subsequently confirmed by a cytochemical study. Thus, the clinical diagnosis was established over a short period of time. This was of importance given the rising severity of the patient’s condition requiring immediate treatment, the initial hyperleukocytosis and the development of life-threatening complications associated with leukostasis in the lungs and the central nervous system. In the presented case, the clinical manifestations of the underlying disease and the results of flow cytofluorimetry were determining factors in initiating timely specific therapy.

Myeloid Sarcoma of the Orbit Preceding Acute Myeloid Leukaemia: A Diagnostic Challenge

Objective: Myeloid sarcoma is a rare form of acute myeloid leukaemia characterized by extramedullary proliferation of myeloid blasts which can occur as an isolated lesion in any organ. Even rarer it may occur in the orbit as the initial presentation without a leukaemic phase and diagnosis may be challenging when it is not suspected.&amp;nbsp; &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&#x0D; &#x0D; Methods: We report a case of orbital myeloid sarcoma as the initial presentation of acute myeloid leukaemia in an adult who was misdiagnosed and treated as a case of a pseudotumour with resultant significant disease progression and worsening of the clinical condition. There was a lag of four months from the onset of eye mass to the development of acute myeloid leukaemia.&#x0D; &#x0D; Result: Due to patients worsening condition and tumour progression, a repeat biopsy for a second histology opinion at a different facility, immunophenotyping and immunohistochemistry were employed to arrive at the correct diagnosis. Following chemotherapy, the orbital mass reduced markedly and clinical condition improved. The patient was indigent and could not sustain further funding of his treatment because he had already spent much on for management of complications he developed before a definitive diagnosis could be made.&#x0D; &#x0D; Conclusion: Myeloid sarcoma can present as an orbital mass without a leukemic disease. Therefore a high index of suspicion, meticulous examination of biopsy, immunohistochemistry and collaboration between oncologists and ophthalmologists, are required to arrive at an early accurate diagnosis.

Endogenous Retinoid X Receptor Ligands Act As Tumor Suppressors in MLL-AF9 Mouse Leukemia

Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL), but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are ligand-activated transcription factors that influence hematopoietic stem cell self-renewal and differentiation. In normal hematopoiesis, RARA and RXRA are dynamically regulated during myeloid maturation, with highest expression in mature neutrophils. In acute myeloid leukemia (AML) RARA and RXRA have parallel expression among AML subtypes, with the highest expression in M4/M5 myelomonocytic and monocytic subtypes. Using a murine reporter assay, we found that natural RXRA ligands, but not RARA ligands, were present in vivo in primary mouse myelomonocytic leukemia cells (derived with an MLL-AF9 retrovirus) and acted as tumor suppressors. RXR ligands were absent in erythroleukemia (derived with TLS-ERG) or T-cell leukemia (derived with activated Notch1), suggesting the presence of natural RXRA ligands specifically in myelomonocytic leukemia. Moreover, we found that deletion of Rxra and Rxrb was naturally selected in MLL-AF9, but not in Notch1 or TLS-ERG leukemias, and that loss of Rxra and Rxrb accelerated leukemic growth. This suggests that Rxrs act as tumor suppressors, but only when exposed to natural ligands. In MLL-AF9 derived leukemia cells, pharmacologic treatment with single-agent retinoid led to modest growth inhibition due to non-permissive activity of the RARA:RXR heterodimer, whereas concurrent activation of both RARA, with all-trans retinoic acid (a pan-RAR ligand), and RXR, by bexarotene (a pan-RXR ligand), enabled efficient co-repressor release and synergistic leukemic apoptosis. We observed that co-repressors release (SMRT/NCoR) from the RARA:RXRA heterodimer was specifically associated with RARA activation, whereas growth inhibition required RXR binding, and this occurred through apoptosis rather than maturation. Generating a series of RXRA mutant proteins we demonstrated the active contribution of RXRA to the activity of the RARA:RXRA heterodimer, specifically requiring the activation domains (AF1 and AF2) and the ability to recruit co-activator to the RXRA element. Finally, we observed a significant dose-dependent effect of combination ATRA and bexarotene treatment in in vivo in MLL-AF9 leukemic mice with a striking reduction in the tumor burden of treated mice compared to the control cohort. These data provide a strategy for clinical retinoid therapies in leukemias beyond acute promyelocytic leukemia and provide a mechanism for integrating retinoid therapy into future clinical trials. Disclosures No relevant conflicts of interest to declare.

Transformation Mechanisms of the Nfia-ETO2 Fusion Gene Associated with Pediatric Pure Acute Erythroleukemia

Pure erythroleukemia (PEL) is a very aggressive, but poorly understood form of acute myeloid leukemia characterized by malignant accumulation of erythroid progenitor cells. A novel t(1;16)(p31;q24) chromosomal translocation leading to expression of a fusion between the nuclear factor I A (NFIA) and the ETO2 transcriptional co-regulator (a.k.a. CBFA2T3 or MTG16) has been identified in pediatric patients with PEL. Based on the function of the fusion partners, we hypothesized that NFIA-ETO2 (N-E) might initiate PEL by interfering with erythroid differentiation. To investigate its function, we cloned a full-length ORF and retrovirally expressed N-E in primary mouse bone marrow (BM)- and fetal liver (FL)-derived erythroblasts (EB). N-E expression significantly increased proliferation and blocked differentiation of EB. N-E expressing BM-derived hematopoietic stem and progenitor cells (HSPC) could be plated in erythropoietin (EPO)-containing methylcellulose (MC) for up to 3 rounds. Expression of N-E deletion mutants lacking the NFIA DNA-binding, the ETO2 NHR2 or NHR4 (ΔNHR4) transcriptional repression domains were unable to block erythroid differentiation. Notably, interfering with the ETO2-NHR2 oligomerization domain by overexpressing a competing peptide overcame the N-E-mediated differentiation block. Transplantation of N-E-expressing BM-derived HSPC into irradiated syngenic mice did not induce any disease suggesting the need of genetic cooperation. As TP53 gain-of-function (GOF) mutations are molecular hallmarks of PEL, we explored functional cooperation by using a conditional TP53R248Q allele. Interestingly, the TP53 status did not affect EB in vitro proliferation or differentiation. However, N-E expression increased proliferation of TP53R248Q+ EB and resulted in the formation of abnormal round and dense colonies in MC that could be serially propagated. In addition, transplantation of N-E-expressing TP53R248Q+ EB into irradiated recipients induced a transplantable PEL-like disease after a median latency of 4 months. Symptomatic mice presented with anemia, thrombocytopenia, multi-organ tumor cell infiltration and increased white blood cell counts. To better understand the molecular mechanism, we compared the gene expression signatures before and 24 hours after induced differentiation of FL-derived EB expressing WT or the inactive ΔNHR4 N-E mutant, in presence or absence of TP53R248Q. Principal component analysis (PCA) revealed a clear separation between the transcriptomes of WT EB expressing either the active or the inactive ΔNHR4 N-E (PC1:54.7%) and by their erythroid differentiation stage (PC2:9.07%). Overall, we observed 3753 (FDR&amp;lt;0.05, logFC&amp;gt;1.5) differentially expressed genes. Many of the significantly higher expressed genes (2313/3753) were related to hematopoietic stemness (GSEAs, p&amp;lt;0.001). Almost 10% of the significantly lower expressed genes (92/1440) were linked to the erythroid lineage development and to erythropoietic targets of NFIA or the erythroid master regulator GATA1. Interestingly, we also found reduced expression of genes encoding for ETO2-interacting transcription factors including TAL1 and KLF1. Despite a critical role on disease progression, PCA showed only minimal changes in the N-E expression signature in presence or absence of TP53R248Q with only 12 genes differently expressed (FDR&amp;lt;0.05, logFC&amp;gt;1). These genes were previously shown to be oncogenic mediators of TP53-GOF mutations, related to metabolism and transcriptional regulation. Interestingly, the signature of differentially expressed genes in N-E transformed FL-derived EB were significantly differentially expressed in tumor cells from pediatric but not adult PEL patients (p=0.00045), indicating the pediatric origin of the fusion. Collectively, we found that the PEL-associated N-E fusion blocks erythroid differentiation, and cooperates with a TP53-GOF mutation to induce a PEL-like disease in mice that phenocopies the human disease. Mechanistically, its activity seems to correlate with repression of erythroid regulatory genes controlled by the fusion partners NFIA, ETO2, and the erythroid master regulator GATA1. Disclosures No relevant conflicts of interest to declare.

Hispanic Race Is Associated with Increased Risk of Severe Sepsis in 1913 Hospitalizations of Children with AML and Infection

Hispanic Race Is Associated with Increased Risk of Severe Sepsis in 1913 Hospitalizations of Children with AML and Infection