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Background: Isocitrate dehydrogenase enzymes catalyze the conversion of isocitrate to alpha-ketoglutarate. Mutated IDH enzymes generate the "oncometabolite" 2-hydroxyglutarate (2-HG), which inhibits TET2 function. IDH2 mutations have been reported in 9% to 19% of acute myeloid leukemia (AML) cases. Inhibition of the mutant IDH2 enzyme led to decreased 2-HG levels and induced myeloid differentiation in IDH2 mutant AML. Enasidenib, a small-molecule inhibitor of mutant IDH2, was approved in 2017, after a successful phase I/II trial showing an overall response rate (ORR) of 40.3% in relapsed/refractory (R/R) disease, with 19.3% of patients achieving complete remission (CR). A Phase III trial is currently ongoing. It targets the mutant IDH2 variants R140Q, R172S, and R172K; at an approved dose of 100 mg oral daily dose. At higher doses the drug was less tolerated, however, few subjects received dose modification at the 650 mg daily dose group; these events did not qualify as a dose-limiting toxicity (DLT). In this descriptive study, we are reporting a case series of R/R AML, with an IDH2 mutation, that are treated with escalated dose enasidenib. Method: This case series is based on retrospective observations of patients with R/R IDH2 mutant AML since January 2017, who are treated with enasidenib. We are reporting two cases treated with an escalated dose of 200 mg daily. We included patients with intermediate to poor-risk AML, who received at least one prior line of therapy, started on standard dose enasidenib (100 mg daily) for a minimum of 6 months and followed until disease relapse or death. We excluded patients who are primarily resistant to enasidenib (AML risk stratification and response evaluation by ELN-Leukemia NET. IDH2 mutations were identified by a local diagnostic laboratory that is regulated under CLIA through PCR which is validated to detect 10% or more mutant allele frequency. To quantify IDH2 variant frequency, confirmatory testing was performed via ARUP next-generation sequencing panel (NGS). Results: Here we report the descriptive outcomes of 2 cases from a single institution, who were treated with escalated dose enasidenib (200 mg oral daily) for R/R AML with an IDH2 mutation. The first case describes a 65-year-old female diagnosed with poor-risk AML in the setting of pancytopenia with bone marrow evaluation consistent with the background of erythroid and granulocytic dysplasia, with normal karyotype analysis. The patient has treated with induction chemotherapy and 2 cycles consolidation that was complicated with bacterial infections, prolonged hospitalization, and profound deconditioning led to stopping treatment. After 18 months of surveillance, repeat bone marrow evaluation for new-onset pancytopenia revealed relapsed AML with evidence of IDH2 mutation at codon 140, with a variant frequency of 36.9% by NGS. The case was started on enasidenib at 100 mg oral daily dose. After 6 months of therapy, the patient continued to be cytopenic, repeated bone marrow evaluation showed residual AML with IDH2 variant frequency of 27.6%. The decision was made to increase the enasidenib dose to 200 mg daily. With follow up for another 5 months on the escalated dose, the patient achieved hematologic complete response (hCR) with normalization of peripheral platelet count and ANC. [Figures 1-2] The patient maintained hCR for another 5 months on 200 mg enasidenib daily until she died from COVID-19 infection. The second case describes a 59-year-old female with poor-risk AML in the setting of leukocytosis, with bone marrow evaluation consistent with the background of megakaryocytic dysplasia and karyotype analysis positive for monosomy 7. The patient was treated with induction chemotherapy and failed to respond. Repeat bone marrow evaluation showed primary refractory disease with positive IDH2 mutation at codon 140 with a variant frequency of 40.7%. Treatment with 100 mg enasidenib was started. The patient maintained a partial response for 6 months, then peripheral blasts counts started to gradually increase. The decision was made to increase enasidenib dose to 200 mg daily, which improved peripheral blasts within one month of escalated dose therapy. [Figure 3] The patient maintained a response to therapy for another 3 months before she relapsed. Conclusion: Our limited data showed that initial response to standard dose enasidenib could potentially be optimized by dose escalation to 200 mg daily. Disclosures No relevant conflicts of interest to declare.

Background BRCA1-associated protein (BAP1) is a tumor suppressor whose loss is associated with various malignancies. The primary cilium is an organelle involved in signal transduction and cell cycle progression. Primary cilia have been shown to be absent in melanoma but retained to some extent in melanocytic nevi, and the severity of dysplasia influences the degree of cilia loss. Additionally, studies have revealed roles for BAP1 in centrosome and mitotic spindle formation. Because the primary cilium is nucleated on the mother centriole, we examined the connection between the presence of primary cilia and the formation of centrosomes in BAP1-inactivated melanocytic tumors (BIMTs). Methods We evaluated the cilia and centrosomes in eleven BIMTs and five conventional melanocytic nevi using immunofluorescence staining of acetylated alpha-tubulin and gamma tubulin. Results We found that, compared to nevi, BIMTs demonstrate loss of primary cilia and amplification of centrosomes. Occasional nevi also showed increased centrioles; however, these foci of amplification were more likely to be ciliated than those in BIMTs. Conclusions Although centrosome amplification does not absolutely correlate with loss of primary cilia in melanocytic neoplasms, absence of BAP1 exacerbates the phenotype. Moreover, aberrant centrosome and cilia formation are likely critical in the pathogenesis of other BAP1-inactivated tumors. This article is protected by copyright. All rights reserved.

While years of investigation have elucidated many aspects of embryonic stem cell (ESC) regulation, the contributions of post-transcriptional and translational mechanisms to the pluripotency network remain largely unexplored. In particular, little is known in ESCs about the function of RNA binding proteins (RBPs), the protein agents of post-transcriptional regulation. We performed an unbiased RNAi screen of RBPs in an ESC differentiation assay and identified two related genes, NF45 (Ilf2) and NF90/NF110 (Ilf3), whose knockdown promoted differentiation to an epiblast-like state. Characterization of NF45 KO, NF90 + NF110 KO, and NF110 KO ESCs showed that loss of NF45 or NF90 + NF110 impaired ESC proliferation and led to dysregulated differentiation down embryonic lineages. Additionally, we found that NF45 and NF90/NF110 physically interact and influence the expression of each other at different levels of regulation. Globally across the transcriptome, NF45 KO ESCs and NF90 + NF110 KO ESCs show similar expression changes. Moreover, NF90 + NF110 RNA immunoprecipitation (RIP)-seq in ESCs suggested that NF90/NF110 directly regulate proliferation, differentiation, and RNA-processing genes. Our data support a model in which NF45, NF90, and NF110 operate in feedback loops that enable them, through both overlapping and independent targets, to help balance the push and pull of pluripotency and differentiation cues.

Establishment, maintenance, and exit from pluripotency require precise coordination of a cell's molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells.

Ectopic expression of specific factors such as Oct4, Sox2, and Klf4 (OSK) is sufficient to reprogram somatic cells into induced pluripotent stem cells (iPSCs). In this study, we examine the paths taken by cells during the reprogramming process by following the transcriptional activation of two pluripotent miRNA clusters (mir-290 and mir-302) in individual cells in vivo and in vitro with knockin reporters. During embryonic development and embryonic stem cell differentiation, all cells sequentially expressed mir-290 and mir-302. In contrast, during OSK-induced reprogramming, cells activated the miRNA loci in a stochastic, nonordered manner. However, the addition of Sall4 to the OSK cocktail led to a consistent reverse sequence of locus activation (mir-302 then mir-290) and increased reprogramming efficiency. These results demonstrate that cells can follow multiple paths during the late stages of reprogramming, and that the trajectory of any individual cell is strongly influenced by the combination of factors introduced. © 2014 Elsevier Inc.