Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL.

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

Ikaros inhibits megakaryopoiesis through functional interaction with GATA-1 and NOTCH signaling.

The transcription factor Ikaros regulates the development of hematopoietic cells. Ikaros-deficient animals fail to develop B cells and display a T-cell malignancy, which is correlated with altered Notch signaling. Recently, loss of Ikaros was associated with progression of myeloproliferative neoplasms to acute myeloid leukemia and increasing evidence shows that Ikaros is also critical for the regulation of myeloid development. Previous studies showed that Ikaros-deficient mice have increased megakaryopoiesis, but the molecular mechanism of this phenomenon remains unknown. Here, we show that Ikaros overexpression decreases NOTCH-induced megakaryocytic specification, and represses expression of several megakaryocytic genes including GATA-1 to block differentiation and terminal maturation. We also demonstrate that Ikaros expression is differentially regulated by GATA-2 and GATA-1 during megakaryocytic differentiation and reveal that the combined loss of Ikzf1 and Gata1 leads to synthetic lethality in vivo associated with prominent defects in erythroid cells and an expansion of megakaryocyte progenitors. Taken together, our observations demonstrate an important functional interplay between Ikaros, GATA factors, and the NOTCH signaling pathway in specification and homeostasis of the megakaryocyte lineage.

HMGA2 expression defines a subset of human AML with immature transcriptional signature and vulnerability to G2/M inhibition.

High-mobility group AT-hook 2 (HMGA2) is a nonhistone chromatin-binding protein that is normally expressed in stem cells of various tissues and aberrantly detected in several tumor types. We recently observed that one-fourth of human acute myeloid leukemia (AML) specimens express HMGA2, which associates with a very poor prognosis. We present results indicating that HMGA2+ AMLs share a distinct transcriptional signature representing an immature phenotype. Using single-cell analyses, we showed that HMGA2 is expressed in CD34+ subsets of stem cells and early progenitors, whether normal or derived from AML specimens. Of interest, we found that one of the strongest gene expression signatures associated with HMGA2 in AML is the upregulation of G2/M checkpoint genes. Whole-genome CRISPR/Cas9 screening in HMGA2 overexpressing cells further revealed a synthetic lethal interaction with several G2/M checkpoint genes. Accordingly, small molecules that target G2/M proteins were preferentially active in vitro and in vivo on HMGA2+ AML specimens. Together, our findings suggest that HMGA2 is a key functional determinant in AML and is associated with stem cell features, G2/M status, and related drug sensitivity.

Genetic characterization of ABT-199 sensitivity in human AML.

Acute myeloid leukemias (AML) with mutations in the NPM1 gene (NPM1c+) represent a large AML subgroup with varying response to conventional treatment, highlighting the need to develop targeted therapeutic strategies for this disease. We screened a library of clinical drugs on a cohort of primary human AML specimens and identified the BCL2 inhibitor ABT-199 as a selective agent against NPM1c+ AML. Mutational analysis of ABT-199-sensitive and -resistant specimens identified mutations in NPM1, RAD21, and IDH1/IDH2 as predictors of ABT-199 sensitivity. Comparative transcriptome analysis further uncovered BCL2A1 as a potential mediator of ABT-199 resistance in AML. In line with our observation that RAD21 mutation confers sensitivity to ABT-199, we provide functional evidence that reducing RAD21 levels can sensitize AML cells to BCL2 inhibition. Moreover, we demonstrate that ABT-199 is able to produce selective anti-AML activity in vivo toward AML with mutations associated with compound sensitivity in PDX models. Overall, this study delineates the contribution of several genetic events to the response to ABT-199 and provides a rationale for the development of targeted therapies for NPM1c+ AML.

Complex karyotype AML displays G2/M signature and hypersensitivity to PLK1 inhibition.

Patients diagnosed with acute myeloid leukemia with complex karyotype (CK AML) have an adverse prognosis using current therapies, especially when accompanied by TP53 alterations. We hereby report the RNA-sequencing analysis of the 68 CK AML samples included in the Leucegene 415 patient cohort. We confirm the frequent occurrence of TP53 alterations in this subgroup and further characterize the allele expression profile and transcript alterations of this gene. We also document that the RAS pathway (N/KRAS, NF1, PTPN11, BRAF) is frequently altered in this disease. Targeted chemical interrogation of genetically characterized primary CK AML samples identifies polo-like kinase 1 (PLK1) inhibitors as the most selective agents for this disease subgroup. TP53 status did not alter sensitivity to PLK1 inhibitors. Interestingly, CK AML specimens display a G2/M transcriptomic signature that includes higher expression levels of PLK1 and correlates with PLK1 inhibition sensitivity. Together, our results highlight vulnerability in CK AML. In line with these in vitro data, volasertib shows a strong anti-AML activity in xenotransplantation mouse models of human adverse AML. Considering that PLK1 inhibitors are currently being investigated clinically in AML and myelodysplastic syndromes, our results provide a new rationale for PLK1-directed therapy in patients with adverse cytogenetic AML.

Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of OXPHOS Dependency in Acute Myeloid Leukemia.

To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34+ hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML.

ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia.

Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.

Design, Synthesis, and Evaluation of Triazole Derivatives That Induce Nrf2 Dependent Gene Products and Inhibit the Keap1-Nrf2 Protein-Protein Interaction.

The transcription factor Nrf2 regulates the expression of a large network of cytoprotective and metabolic enzymes and proteins. Compounds that directly and reversibly inhibit the interaction between Nrf2 and its main negative regulator Keap1 are potential pharmacological agents for a range of disease types including neurodegenerative conditions and cancer. We describe the development of a series of 1,4-diphenyl-1,2,3-triazole compounds that inhibit the Nrf2-Keap1 protein-protein interaction (PPI) in vitro and in live cells and up-regulate the expression of Nrf2-dependent gene products.

Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models.

Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non-Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors.