P. G. Pelicci

Arsenic trioxide (ATO) and MEK1 inhibition synergize to induce apoptosis in acute promyelocytic leukemia cells

Recent studies suggest that components of the prosurvival signal transduction pathways involving the Ras-mitogen-activated protein kinase (MAPK) can confer an aggressive, apoptosis-resistant phenotype to leukemia cells. In this study, we report that acute promyelocytic leukemia (APL) cells exploit the Ras-MAPK activation pathway to phosphorylate at Ser112 and to inactivate the proapoptotic protein Bad, delaying arsenic trioxide (ATO)-induced apoptosis. Both in APL cell line NB4 and in APL primary blasts, the inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) and Bad phosphorylation by MEK1 inhibitors enhanced apoptosis in ATO-treated cells. We isolated an arsenic-resistant NB4 subline (NB4-AsR), which showed stronger ERK1/2 activity (2.7-fold increase) and Bad phosphorylation (2.4-fold increase) compared to parental NB4 cells in response to ATO treatment. Upon ATO exposure, both NB4 and NB4-AsR cell lines doubled protein levels of the death antagonist Bcl-xL, but the amount of free Bcl-xL that did not heterodimerize with Bad was 1.8-fold greater in NB4-AsR than in the parental line. MEK1 inhibitors dephosphorylated Bad and inhibited the ATO-induced increase of Bcl-xL, overcoming ATO resistance in NB4-AsR. These results may provide a rationale to develop combined or sequential MEK1 inhibitors plus ATO therapy in this clinical setting.

The PML/RARalpha fusion protein inhibits tumor necrosis factor-alpha-induced apoptosis in U937 cells and acute promyelocytic leukemia blasts.

We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.

The self-association coiled-coil domain of PML is sufficient for the oncogenic conversion of the retinoic acid receptor (RAR) alpha

In acute promyelocytic leukemia (APL) the retinoic acid receptor alpha (RARα) becomes an oncogene through the fusion with several partners, mostly with promyelocytic leukemia protein (PML), all of which have in common the presence of a self-association domain. The new fusion proteins, therefore, differently from the wild-type RARα, which forms only heterodimers with retinoic X receptor alpha, are also able to homo-oligomerize. The presence of such a domain has been suggested to be crucial for the leukemogenic potential of the chimeric proteins found in APL blasts. Whether or not any self-association domain is sufficient to bestow a leukemogenic activity on RARα is still under investigation. In this work, we address this question using two different X-RARα chimeras, where X represents the coiled-coil domain of PML (CC-RARα) or the oligomerization portion of the yeast transcription factor GCN4 (GCN4–RARα). We demonstrate that in vitro both proteins have transforming potential, and recapitulate the main PML–RARα biological properties, but CC-RARα is uniquely able to disrupt PML nuclear bodies. Indeed, in vivo only the CC-RARα chimera induces efficiently APL in a murine transplantation model. Thus, the PML CC domain represents the minimal structural determinant indispensable to transform RARα into an oncogenic protein.

Acute promyelocytic leukemias share cooperative mutations with other myeloid-leukemia subgroups.

Correction to: Blood Cancer Journal (2013) 3, e147; doi: 10.1038/bcj.2013.46; published online 13 September 2013

A redundant oncogenic potential of the retinoic receptor (RAR) alpha, beta and gamma isoforms in acute promyelocytic leukemia.

Alterations of the retinoic acid receptor (RAR)α locus are found in 100% of acute promyelocytic leukemia patients, where chromosomal translocations generate the promyelocytic leukemia (PML)-RARα chimeric protein. Here, we have investigated the biological properties of the other RAR isoforms (RARβ and RARγ), through the generation and characterization of artificial PML-RAR‘x’ fusion proteins. Surprisingly, we found that all of the RAR isoforms share an identical oncogenic potential in vitro, thus implying that the selection of the RARα locus in leukemia patients must occur – rather than through functional differences among the various RAR isoforms–as the consequence of the nuclear architecture of the different RAR loci.

The Combination of the PARP Inhibitor Rucaparib and 5FU Is an Effective Strategy for Treating Acute Leukemias

The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most patients, drawing attention to the need of new therapeutic strategies. In the last decade the anticancer potential of poly ADP-ribose polymerase (PARP) inhibitors became apparent and now several PARP inhibitors are being developed to treat various malignancies. So far, the usage of PARP inhibitors has been mainly focused on the treatment of solid tumors and not too much about their efficacy on leukemias is known. In this study we test, for the first time on leukemic cells, a combined therapy that associates the conventional chemotherapeutic agent fluorouracil (5FU), used as a source of DNA damage, and a PARP inhibitor, rucaparib. We demonstrate the efficacy and the specificity of this combined therapy in killing both acute myeloid leukemia and acute lymphoid leukemia cells in vitro and in vivo. We clearly show that the inhibition of DNA repair induced by rucaparib is synthetic lethal with the DNA damage caused by 5FU in leukemic cells. Therefore, we propose a new therapeutic strategy able to enhance the cytotoxic effect of DNA-damaging agents in leukemia cells via inhibiting the repair of damaged DNA. Mol Cancer Ther; 14(4); 889–98. ©2015 AACR.

Redundant function of retinoic acid receptor isoforms in leukemogenesis unravels a prominent function of genome topology and architecture in the selection of mutagenic events in cancer.

Retinoic acid receptors (RARs) are members of a much larger group of ligand-dependent transcription factors, the so-called nuclear receptors. In mammals, there are three distinct RAR subtypes: RAR, RAR and RAR.1 RARs share a high sequence homology, but show distinct transcriptional properties and expression pattern, suggesting similar but not necessarily overlapping functions. In haematopoietic differentiation, for example, the function of RAR is uniquely required to maintain the stem cell compartment, whereas RAR seems to work primarily at later stages, favouring differentiation.2 In acute promyelocytic leukaemia (APL), chromosomal translocations involve in 100% of cases the -subtype of the RAR gene (RAR), on chromosome 17. These translocations lead to the synthesis of chimeric proteins: in 95% of the cases, the translocation partner is the PML (promyelocytic leukaemia) gene on chromosome 15, leading to the formation of PML–RAR fusion gene.3 Both in vitro and in vivo studies have well defined the oncogenic roles of PML–RAR fusion protein in the pathogenesis of APL, acting as an altered RAR.4 In our previous work, we studied the transcriptional and biological properties of chimeric constructs where PML was fused to RAR or RAR to generate the PML–RAR and PML–RAR fusion proteins (which do not exist in nature). Both chimeric proteins demonstrated similar properties to PML–RAR in vitro.5 Here, we extended this analysis to determine directly whether PML–RAR (as a test case), like PML–RAR, is able to induce APL.

PML-RARA-associated cooperating mutations belong to a transcriptional network that is deregulated in myeloid leukemias

It has been shown that individual acute myeloid leukemia (AML) patients are characterized by one of few initiating DNA mutations and 5–10 cooperating mutations not yet defined among hundreds identified by massive sequencing of AML genomes. We report an in vivo insertional-mutagenesis screen for genes cooperating with one AML initiating mutations (PML-RARA, oncogene of acute promyelocytic leukemia, APL), which allowed identification of hundreds of genetic cooperators. The cooperators are mutated at low frequency in APL or AML patients but are always abnormally expressed in a cohort of 182 APLs and AMLs analyzed. These deregulations appear non-randomly distributed and present in all samples, regardless of their associated genomic mutations. Reverse-engineering approaches showed that these cooperators belong to a single transcriptional gene network, enriched in genes mutated in AMLs, where perturbation of single genes modifies expression of others. Their gene-ontology analysis showed enrichment of genes directly involved in cell proliferation control. Therefore, the pool of PML-RARA cooperating mutations appears large and heterogeneous, but functionally equivalent and deregulated in the majority of APLs and AMLs. Our data suggest that the high heterogeneity of DNA mutations in APLs and AMLs can be reduced to patterns of gene expression deregulation of a single ‘mutated’ gene network.

Erratum: Arsenic trioxide (ATO) and MEK1 inhibition synergize to induce apoptosis in acute promyelocytic leukemia cells (Leukemia (2005) vol. 19 (234-244) 10.1038/sj.leu.2403585)

Correction to: Leukemia (2005), 19, 234–244. doi:10.1038/sj.leu.2403585 The authors have identified that the name of the coauthor P Pelicci and his affiliation are incomplete. The correct name and affiliation are reproduced here: PG Pelicci Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.