Blockade of deubiquitinase YOD1 degrades oncogenic PML/RARα and eradicates acute promyelocytic leukemia cells.

In most acute promyelocytic leukemia (APL) cells, promyelocytic leukemia (PML) fuses to retinoic acid receptor α (RARα) due to chromosomal translocation, thus generating PML/RARα oncoprotein, which is a relatively stable oncoprotein for degradation in APL. Elucidating the mechanism regulating the stability of PML/RARα may help to degrade PML/RARα and eradicate APL cells. Here, we describe a deubiquitinase (DUB)-involved regulatory mechanism for the maintenance of PML/RARα stability and develop a novel pharmacological approach to degrading PML/RARα by inhibiting DUB. We utilized a DUB siRNA library to identify the ovarian tumor protease (OTU) family member deubiquitinase YOD1 as a critical DUB of PML/RARα. Suppression of YOD1 promoted the degradation of PML/RARα, thus inhibiting APL cells and prolonging the survival time of APL cell-bearing mice. Subsequent phenotypic screening of small molecules allowed us to identify ubiquitin isopeptidase inhibitor I (G5) as the first YOD1 pharmacological inhibitor. As expected, G5 notably degraded PML/RARα protein and eradicated APL, particularly drug-resistant APL cells. Importantly, G5 also showed a strong killing effect on primary patient-derived APL blasts. Overall, our study not only reveals the DUB-involved regulatory mechanism on PML/RARα stability and validates YOD1 as a potential therapeutic target for APL, but also identifies G5 as a YOD1 inhibitor and a promising candidate for APL, particularly drug-resistant APL treatment.

All-trans Retinoic Acid-Induced Isolated Myocarditis: A Case Report and Review of the Literature.

All-trans retinoic acid (ATRA) is the mainstay of treatment in patients with acute promyelocytic leukemia. Despite being effective, it can lead to cardiac complications either as a component of ATRA syndrome or an isolated form denominated as ATRA-induced isolated perimyocarditis. We present a case of this complication and review the literature. (Level of Difficulty: Intermediate.).

Secondary clonal hematologic neoplasia following successful therapy for acute promyelocytic leukemia (APL): A report of two cases and review of the literature.

Although rare, secondary clonal hematologic neoplasia may occur after successful therapy for acute promyelocytic leukemia (APL). These secondary clonal events may be considered therapy-related, but may also be due to an underlying background of clonal hematopoiesis from which both malignancies may develop. In this manuscript, we describe two patients with secondary clones after APL therapy characterized in one patient by deletion of chromosome 11q23 and, in the other, by monosomy of chromosome 7, and also provide a review of all secondary clonal disorders described after APL therapy. We suggest that since most reports identify karyotypic abnormalities not typically associated with chemotherapy, there may be another mechanism underlying secondary clonal development after complete response to initial APL therapy.

The AhR agonist VAF347 augments retinoic acid-induced differentiation in leukemia cells.

In binary cell-fate decisions, driving one lineage and suppressing the other are conjoined. We have previously reported that aryl hydrocarbon receptor (AhR) promotes retinoic acid (RA)-induced granulocytic differentiation of lineage bipotent HL-60 myeloblastic leukemia cells. VAF347, an AhR agonist, impairs the development of CD14(+)CD11b(+) monocytes from granulo-monocytic (GM) stage precursors. We thus hypothesized that VAF347 propels RA-induced granulocytic differentiation and impairs D3-induced monocytic differentiation of HL-60 cells. Our results show that VAF347 enhanced RA-induced cell cycle arrest, CD11b integrin expression and neutrophil respiratory burst. Granulocytic differentiation is known to be driven by MAPK signaling events regulated by Fgr and Lyn Src-family kinases, the CD38 cell membrane receptor, the Vav1 GEF, the c-Cbl adaptor, as well as AhR, all of which are embodied in a putative signalsome. We found that the VAF347 AhR ligand regulates the signalsome. VAF347 augments RA-induced expression of AhR, Lyn, Vav1, and c-Cbl as well as p47(phox). Several interactions of partners in the signalsome appear to be enhanced: Fgr interaction with c-Cbl, CD38, and with pS259c-Raf and AhR interaction with c-Cbl and Lyn. Thus, we report that, while VAF347 impedes monocytic differentiation induced by 1,25-dihydroxyvitamin D3, VAF347 promotes RA-induced differentiation. This effect seems to involve but not to be limited to Lyn, Vav1, c-Cbl, AhR, and Fgr.

Induced myelomonocytic differentiation in leukemia cells is accompanied by noncanonical transcription factor expression.

Transcription factors that drive non-neoplastic myelomonocytic differentiation are well characterized but have not been systematically analyzed in the leukemic context. We investigated widely used, patient-derived myeloid leukemia cell lines with proclivity for differentiation into granulocytes by retinoic acid (RA) and/or monocytes by 1,25-dihyrdroxyvitamin D3 (D3). Using K562 (FAB M1), HL60 (FAB M2), RA-resistant HL60 sublines, NB4 (FAB M3), and U937 (FAB M5), we correlated nuclear transcription factor expression to immunophenotype, G1/G0 cell cycle arrest and functional inducible oxidative metabolism. We found that myelomonocytic transcription factors are aberrantly expressed in these cell lines. Monocytic-lineage factor EGR1 was not induced by D3 (the monocytic inducer) but instead by RA (the granulocytic inducer) in lineage bipotent myeloblastic HL60. In promyelocytic NB4 cells, EGR1 levels were increased by D3, while Gfi-1 expression (which promotes the granulocytic lineage) was upregulated during D3-induced monocytic differentiation in HL60, and by RA treatment in monocytic U937 cells. Furthermore, RARα and VDR expression were not strongly correlated to differentiation. In response to different differentiation inducers, U937 exhibited the most distinct transcription factor expression profile, while similarly mature NB4 and HL60 were better coupled. Overall, the differentiation induction agents RA and D3 elicited cell-specific responses across these common FAB M1-M5 cell lines.

Neurologic aspects of lymphoma and leukemias.

The lymphomas and leukemias are a heterogenous group of hematologic malignancies with protean manifestations. Neurologic sequelae of the diseases have been recognized since the time the conditions were first described in the mid-1800s. Although our understanding of the various presentations of these blood disorders evolved along with our knowledge of malignancies, accurate diagnosis can still be difficult. It is critical for neurologists to have a high index of clinical suspicion to appropriately recognize their heralding features. This review's focus is the relevant clinical neurologic features and diagnostic studies that identify leukemias and lymphomas affecting the nervous system.

The oncogene EVI1 enhances transcriptional and biological responses of human myeloid cells to all-trans retinoic acid.

The product of the ecotropic virus integration site 1 (EVI1) gene, whose overexpression is associated with a poor prognosis in myeloid leukemias and some epithelial tumors, regulates gene transcription both through direct DNA binding and through modulation of the activity of other sequence specific transcription factors. Previous results from our laboratory have shown that EVI1 influenced transcription regulation in response to the myeloid differentiation inducing agent, all-trans retinoic acid (ATRA), in a dual manner: it enhanced ATRA induced transcription of the RARß gene, but repressed the ATRA induction of the EVI1 gene itself. In the present study, we asked whether EVI1 would modulate the ATRA regulation of a larger number of genes, as well as biological responses to this agent, in human myeloid cells. U937 and HL-60 cells ectopically expressing EVI1 through retroviral transduction were subjected to microarray based gene expression analysis, and to assays measuring cellular proliferation, differentiation, and apoptosis. These experiments showed that EVI1 modulated the ATRA response of several dozens of genes, and in fact reinforced it in the vast majority of cases. A particularly strong synergy between EVI1 and ATRA was observed for GDF15, which codes for a member of the TGF-ß superfamily of cytokines. In line with the gene expression results, EVI1 enhanced cell cycle arrest, differentiation, and apoptosis in response to ATRA, and knockdown of GDF15 counteracted some of these effects. The potential clinical implications of these findings are discussed.

Importance of assessing cytogenetic and molecular risk factors in acute myeloid leukemia therapy.

Acute myeloid leukemia (AML) is a heterogeneous disease in clinical presentation, outcome and therapeutic response. Cytogenetic and molecular characteristics are important prognostic indicators allowing the identification of distinct subtypes of AML, prognostic stratification and risk-adapted treatment. We present our experience during 5 years, in which we treated 245 patients with AML, of which we could genetically characterize 48 cases (26 females, 22 males) with a median age of 52 years. Cytogenetic analysis was performed by GTG banding on cultures of marrow cells treated with colcemid. Molecular analysis used RT-PCR performed on ABI 9700 platform in order to identify the following fusion genes: E2A-PBX1, TEL-AML1, AML1-ETO, PML-RARα, MLL-AF4, CBFC-MYH11, BCR-ABL, SIL-TAL, and MLL-AF9as well as mutations in Flt3, NPM1, WT1 genes. Fourteen patients were older than 60 years. In 12 we performed cytogenetic analysis showing 5 cases with complex karyotype, 2 normal karyotypes, 1 case of del(21), del (9), 11q- and t(3;15) respectively as well as 2 unevaluable karyotypes. These anomalies were associated with a high incidence of secondary AMLs (10/14) and with a low remission (CR) rate (5/14). Out of the 35 patients younger than 60 years, 25 were evaluated by cytogenetics showing a high incidence of favorable cytogenetic changes: 6 anomalies of chromosome 16 (5 inv (16) and 1 t (16; 16)), 3 t (15; 17), 3 cases of t (8; 21) of which 2 with additional abnormalities, 7 normal karyotypes and 1 case of 7q-, -y,-3 and respectively -8 associated with +18. In 25 cases molecular analysis was performed showing alterations in 21 patients: 6 cases with AML/ETO, 3 PML/RAR, 7 Flt3 mutations (2 associated with NPM1 mutation) as well as 1 case of isolated mutation of NPM1 and respectively WT1. CR rate was of 28/35. All cases with t (15; 17) and PML/RAR as well all cases with t (8; 21) and/or AML/ETO achieved CR. Out of the 7 cases with Flt3 mutations only 4 achieved CR including the 2 cases with associated NPM1 mutations. In our experience, genetic characteristics correlate with other prognostic markers such as age and secondary leukemia; "favorable" genetic anomalies were associated with a high CR rate; association of t (8; 21) with additional abnormalities did not influence CR rate.