Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 and CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and therefore be candidates for experimental therapies. In addition, there have been few other molecular pathways identified aside from the Ras/MAPK pathway to serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia in order to expand our knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, gene splicing, the polycomb repressive complex 2 (PRC2) and transcription. Importantly, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome.

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We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML. (Blood. 2006;108:3477-3483)

IMPORTANCE Standard chemotherapy for first relapse of B-cell acute lymphoblastic leukemia (B-ALL) in children, adolescents, and young adults is associated with high rates of severe toxicities, subsequent relapse, and death, especially for patients with early relapse (high risk) or late relapse with residual disease after reinduction chemotherapy (intermediate risk). Blinatumomab, a bispecific CD3 to CD19 T cell-engaging antibody construct, is efficacious in relapsed/refractory B-ALL and has a favorable toxicity profile.OBJECTIVE To determine whether substituting blinatumomab for intensive chemotherapy in consolidation therapy would improve survival in children, adolescents, and young adults with high-and intermediate-risk first relapse of B-ALL. DESIGN, SETTING, AND PARTICIPANTSThis trial was a randomized phase 3 clinical trial conducted by the Children's Oncology Group at 155 hospitals in the US, Canada, Australia, and New Zealand with enrollment from December 2014 to September 2019 and follow-up until September 30, 2020. Eligible patients included those aged 1 to 30 years with B-ALL first relapse, excluding those with Down syndrome, Philadelphia chromosome-positive ALL, prior hematopoietic stem cell transplant, or prior blinatumomab treatment (n = 669).INTERVENTIONS All patients received a 4-week reinduction chemotherapy course, followed by randomized assignment to receive 2 cycles of blinatumomab (n = 105) or 2 cycles of multiagent chemotherapy (n = 103), each followed by transplant. MAIN OUTCOME AND MEASURESThe primary end point was disease-free survival and the secondary end point was overall survival, both from the time of randomization. The threshold for statistical significance was set at a 1-sided P <.025. RESULTS Among 208 randomized patients (median age, 9 years; 97 [47%] females), 118 (57%) completed the randomized therapy. Randomization was terminated at the recommendation of the data and safety monitoring committee without meeting stopping rules for efficacy or futility; at that point, 80 of 131 planned events occurred. With 2.9 years of median follow-up, 2-year disease-free survival was 54.4% for the blinatumomab group vs 39.0% for the chemotherapy group (hazard ratio for disease progression or mortality, 0.70 [95% CI, 0.47-1.03]); 1-sided P = .03). Two-year overall survival was 71.3% for the blinatumomab group vs 58.4% for the chemotherapy group (hazard ratio for mortality, 0.62 [95% CI, 0.39-0.98]; 1-sided P = .02). Rates of notable serious adverse events included infection (15%), febrile neutropenia (5%), sepsis (2%), and mucositis (1%) for the blinatumomab group and infection (65%), febrile neutropenia (58%), sepsis (27%), and mucositis (28%) for the chemotherapy group.CONCLUSIONS AND RELEVANCE Among children, adolescents, and young adults with high-and intermediate-risk first relapse of B-ALL, postreinduction treatment with blinatumomab compared with chemotherapy, followed by transplant, did not result in a statistically significant difference in disease-free survival. However, study interpret...

IntroductionConstitutive activation of the class III receptor tyrosine kinase, FLT3, plays important roles in leukemogenesis. [1][2][3][4] Internal tandem duplications in the juxtamembrane region (FLT3-ITD) or point mutations in the kinase domain (FLT3-PM) lead to constitutively activated FLT3. [5][6][7] FLT3 is also activated by coexpression of FLT3 ligand (FL) through intracrine, paracrine, and/or autocrine pathways. [8][9][10] The presence of FLT3-ITD mutations is associated with a poor prognosis in acute myelogenous leukemia (AML). 11-14 Activated FLT3 mediates signaling through at least 3 major downstream signaling pathways: signal transducers and activators of transcription (STAT5), PI3K/Akt, and Ras/mitogen-activated protein (MAP) kinase. [15][16][17][18][19][20][21][22][23][24][25] These signaling pathways have overlapping roles in cell differentiation, proliferation, and survival. FLT3 is expressed in most acute leukemias, including 94% of B-lineage acute lymphoblastic leukemia (ALL), 34% of T-lineage ALL, and 89% of AML cases. [26][27][28] These observations strongly suggest FLT3 as a candidate for molecularly targeted therapy.In fact, a number of FLT3 tyrosine kinase inhibitors (TKIs) have been developed. Some of the best studied to date include CEP-701 (lestaurtinib), PKC412, MLN518, SU11248 (sunitinib malate), and AG1295. 23,[29][30][31][32][33] Although these inhibitors vary in their potency and selectivity for FLT3, all are able to induce cytotoxicity in FLT3-expressing cells in vitro and/or in vivo. Furthermore, clinical trials with some of these inhibitors have demonstrated their ability to decrease peripheral blood and bone marrow blast counts in some patients. [34][35][36] CEP-701 is currently being tested on relapsed patients with FLT3 mutant AML in a randomized phase 2 clinical trial in combination with chemotherapy.Although FLT3 inhibitors demonstrate preclinical and clinical activity, they possess a number of limitations. Clinical trials have revealed that FLT3 TKIs used as single agents are able to significantly reduce peripheral blood and bone marrow blasts only in a minority of patients, and the effect is transitory. [34][35][36] This may be due to achieving insufficient levels of FLT3 inhibition in these patients, a lack of dependence of these cells on FLT3 signaling for proliferation and survival, and/or selection of resistant cell populations. Furthermore, most cases of AML and ALL do not express mutant FLT3, and it is unclear to what degree these cells depend on FLT3 signaling for sustaining the leukemic phenotype. At drug concentrations necessary to inhibit FLT3 phosphorylation past a critical threshold required to induce cytotoxicity, a varying spectrum of other kinases are frequently also inhibited, which can lead to toxicities.Even when cells are dependent on FLT3 signaling for survival and proliferation, prolonged exposure to TKIs are likely to select for resistant clones, as has been seen with imatinib mesylate (Gleevec), a TKI targeting BCR-ABL in chronic myelogenous leukemia...

Frameshift mutations in exon 12 of the nucleophosmin gene (NPM1) result in aberrant cytoplasmic localization of the NPM protein (NPMc ؉ ) and occur in 25% to 35% of adult acute myeloid leukemia (AML). In adults with AML, NPMc ؉ has been associated with normal karyotype, FLT3/ITD mutations, high remission induction rates, and improved survival (particularly in patients lacking FLT3/ITD). NPMc ؉ has not been well characterized in childhood AML. This study examines the incidence and clinical significance of NPMc ؉ in 295 children with newly diagnosed AML treated on a large cooperative group clinical trial (POG-9421). We find that NPMc ؉ is relatively uncommon in childhood AML (23 of 295 patients, 8%); and is significantly associated with FLT3/ITD mutations (P ‫؍‬ .046), female sex (P ‫؍‬ .029), older age (P ‫؍‬ .047), and normal cytogenetics (P < .001). There is a favorable impact of NPMc ؉ on survival in children lacking FLT3/ITD (5-year EFS, 69% vs 35%; hazard ratio, 0.39; P ‫؍‬ .051), which is similar in magnitude to the favorable impact of t(8;21) and inv(16). We conclude that NPMc ؉ is relatively rare in childhood AML, particularly in younger children. IntroductionAcute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease that accounts for 15% to 20% of childhood leukemia. Currently, cytogenetic analysis at diagnosis allows for risk stratification of childhood AML into favorable, adverse, and intermediate risk groups. 1 Treatment protocols are, to some extent, risk adapted in an attempt to improve survival and decrease treatment-related toxicity. Unfortunately, prognostic implications have not been reliably established for AML in the intermediate risk category, a group that includes 60% to 70% of childhood AML. In recent years, molecular analysis has identified novel markers with prognostic relevance in this diverse group. For example, cases of AML with internal tandem duplications of fms-like tyrosine kinase-3 (FLT3/ITD) have a poor prognosis. [2][3][4][5] Conversely, cases with a mutation in the transcription factor CCAAT/enhancerbinding protein-␣ (CEBPA) have a more favorable prognosis. 6,7 Mutations in exon 12 of nucleophosmin (NPM1) have been investigated recently as another potentially useful molecular marker in AML. 8 NPM1 encodes a phosphoprotein that is ubiquitously expressed and is highly conserved. The protein is localized primarily to the nucleolus, but shuttles rapidly between the nucleus and cytoplasm in its role as a molecular chaperone. 9 The NPM1 protein has been shown to contribute to many basic cellular processes, including biosynthesis of ribosomes, 10 preventing aggregation of proteins in the nucleolus, 11 and regulating centrosome duplication through cyclin E/cyclin-dependent kinase 2 phosphorylation. 12 Further, it has been found to be a stress-induced regulator of p53 function 13 and is a nucleolar binding partner of ARF, involved in p53-independent cell cycle regulation. 14 Abnormalities in NPM1 have been implicated in the pathogenesis of various hematopoietic ...

The FLT3 receptor tyrosine kinase is highly expressed in most acute leukemias and frequently mutated in acute myeloid leukemia (AML