Search

Standard chemotherapies for diffuse large B-cell lymphoma (DLBCL), based on the induction of exogenous DNA damage and oxidative stress, are often less effective in the presence of increased MYC and BCL-2 levels, especially in the case of double hit (DH) lymphomas harboring rearrangements of the MYC and BCL-2 oncogenes, which enrich for a patient's population characterized by refractoriness to anthracycline-based chemotherapy. Here we hypothesized that adaptive mechanisms to MYC-induced replicative and oxidative stress, consisting in DNA damage response (DDR) activation and BCL-2 overexpression, could represent the biologic basis of the poor prognosis and chemoresistance observed in MYC/BCL-2-positive lymphoma. We first integrated targeted gene expression profiling (T-GEP), fluorescence in situ hybridization (FISH) analysis, and characterization of replicative and oxidative stress biomarkers in two independent DLBCL cohorts. The presence of oxidative DNA damage biomarkers identified a poor prognosis double expresser (DE)-DLBCL subset, characterized by relatively higher BCL-2 gene expression levels and enrichment for DH lymphomas. Based on these findings, we tested therapeutic strategies based on combined DDR and BCL-2 inhibition, confirming efficacy and synergistic interactions in in vitro and in vivo DH-DLBCL models. These data provide the rationale for precision-therapy strategies based on combined DDR and BCL-2 inhibition in DH or DE-DLBCL., (© 2021. The Author(s).)

The E3 ubiquitin ligase (E3) WWP1 is an oncogenic factor implicated in the maintenance of different types of epithelial cancers. The role of WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in haematological neoplasms remains unknown. Acute myeloid leukaemia (AML) is characterized by the expansion of malignant myeloid cells blocked at different stages of differentiation. Here we report that the expression of WWP1 is significantly augmented in a large cohort of primary AML patients and in AML cell lines, compared with haematopoietic cells from healthy donors. We show that WWP1 inactivation severely impairs the growth of primary AML blasts and cell lines in vitro. In vivo, we observed a reduced leukaemogenic potential of WWP1-depleted AML cells upon transplantation into immunocompromised mice. Mechanistically, WWP1 inactivation induces the accumulation of its protein substrate p27 Kip1 , which ultimately contributes to G 0 /G 1 cell cycle arrest of AML blasts. In addition, WWP1 depletion triggers the autophagy signalling and reduces survival of leukaemic cells. Collectively, our findings provide molecular insights into the anti-cancer potential of WWP1 inhibition, suggesting that this E3 is a promising biomarker and druggable target in AML.

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.

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.

Alterations of the retinoic acid receptor (RAR)alpha locus are found in 100% of acute promyelocytic leukemia patients, where chromosomal translocations generate the promyelocytic leukemia (PML)-RARalpha chimeric protein. Here, we have investigated the biological properties of the other RAR isoforms (RARbeta and RARgamma), 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 RARalpha 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.

Six patients with de novo acute myeloid leukemia (AML) and a t(2;3)(p15-21;q26-27) were identified among approximately 1000 cases enrolled in the GIMEMA trial. The t(2;3) was the sole anomaly in three patients, whereas in three cases monosomy 7, trisomy 15 and 22, and trisomy 14 represented additional aberrations. No cryptic chromosome deletions at 5q, 7q, 12p, and 20q were observed. One patient carried a FLT3 D835 mutation; FLT3 internal tandem duplication (ITD) was not detected in three patients tested. Characterization of the translocation breakpoints using a 3q26 BAC contig specific for the PRDM3 locus showed that the breakpoints were located 5' to EVIl as follows: within myelodysplatic syndrome (MDS) intron 1 (# 3), between MDS1 exons 2 and 3 in three patients (# 1, 2, 4) with a 170bp cryptic deletion distal to the breakpoint in one (# 2), and in a more centromeric position spanning from intron 2 to the 5' region of EVI1 (# 6, 5). A set of 2p16-21 BAC probes showed that the breakpoints on chromosome 2p were located within BCL11A in two separate regions (# 1, 4 and # 2-5), within the thyroid adenoma-associated (THADA) gene (# 6) or distal to the ZFP36L2 locus (# 3). Regulatory elements were present in proximity of these breakpoints. RACE PCR studies revealed a chimeric transcript in 1/6 patient analyzed, but no fusion protein. Quantitative PCR showed a 21-58-fold over-expression of the EVIl gene in all cases analyzed. The patients showed dysplasia of at least two myeloid cell lineages in all cases; they had a low-to-normal platelet count and displayed an immature CD34+ CD117+ immunophenotype. Despite intensive chemotherapy and a median age of 43 years (range 36-59), only two patients attained a short-lived response; one patient is alive with active disease at 12 months, five died at 4-14 months. We arrived at the following conclusions: (a) the t(2;3) is a recurrent translocation having an approximate 0.5% incidence in adult AML; (b) breakpoints involve the 5' region of EVIl at 3q26, and the BCL11A, the THADA gene or other regions at 2p16.1-21; (c) cryptic deletions distal to the 3q26 breakpoint may occur in some cases; (d) the juxtaposition of the 5' region of EVIl with regulatory elements normally located on chromosome 2 brings about EVI1 overexpression; (e) clinical outcome in these cases is severe.

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-As(R)), 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-As(R) 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-As(R) than in the parental line. MEK1 inhibitors dephosphorylated Bad and inhibited the ATO-induced increase of Bcl-xL, overcoming ATO resistance in NB4-As(R). These results may provide a rationale to develop combined or sequential MEK1 inhibitors plus ATO therapy in this clinical setting.

MAP kinase/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) kinases are frequently activated in acute myelogenous leukemia (AML), and can have prosurvival function. The purpose of this study was to induce downmodulation of MEK-ERK activation in AML primary blasts in order to detect the effect on cell cycle progression and on the apoptosis of leukemic cells. We investigated 14 cases of AML with high ERK 1/2 activity and four cases with undetectable or very low activity. After 24 h incubation of the AML blasts with high ERK activity using PD98059 (New England BioLabs, Beverly, MA, USA), a selective inhibitor of MEK1 phosphorylation, at concentrations of 20 and 40 microM, we observed a strong decrease in the levels of ERK1/2 activity. A significant decrease of blast cell proliferation compared with untreated controls was found. In contrast, the proliferation of blast cells that expressed low or undetectable levels of ERK activity was not inhibited. Time-course analysis demonstrated that the downmodulation of MEK1/2, ERK1 and ERK2 dual-phosphorylation was evident even after 3 h of treatment with 20 and 40 microM. The cleavage of poly(ADP-ribose) polymerase (PARP), an early sign of apoptosis, appeared after 18 h of PD98059 treatment at concentrations of 20 and 40 microM in eight of the 14 cases. After 24 h of treatment, cleaved PARP appeared in all 14 cases. Time-course analysis of cell cycle progression and apoptosis showed that PD98059 induced a G1-phase accumulation with low or undetectable levels of apoptosis after 24 h incubation; after 48 and 72 h incubation, a significant increase of apoptosis was observed. Thus, the primary effect of ERK downmodulation was a cell cycle arrest followed by the apoptosis of a significant percentage of the leukemic blasts. The preclinical model of leukemia treatment reported in this paper makes further comment with regard to MEK1 inhibition as a useful antileukemic target, and encourages the conducting of in vivo studies and clinical investigations.

Typical acute promyelocytic leukemia (APL) is associated with the t(15;17) translocation, expression of a PML/RARA fusion transcript, and responsiveness to all-trans retinoic acid (ATRA). Rare APL cases implicating the RARA but not the PML gene have been reported. Cases with t(11;17)(q23;q21) which fuses the PLZF and RARA genes do not respond to ATRA. In contrast, cases with t(11;17)(q13;q21) and t(5;17)(q35;q21) which fuse RARA with NuMA and NPM, respectively, were reported to be sensitive to ATRA. We described previously an APL case with an unbalanced t(5;17) implicating RARA but neither PML nor PLZF. Here, we show that in this case: (1) the NPM gene is not involved, as demonstrated by RT-PCR and Southern blot; (2) response to ATRA in vitro is atypical, as demonstrated by morphological and functional maturation assays; and (3) PML nuclear bodies are not disrupted, as evidenced by immunofluorescence staining.

We have analyzed the differentiation program of growth factor-dependent TF-1 erythroleukemia cells as well as clones with inducible expression of the APL-specific PML/RARalpha protein. We have shown that TF-1 cells may be induced to megakaryocytic differentiation by phorbol ester (phorbol dibutyrate, PDB) addition, particularly when combined with thrombopoietin (Tpo). RT-PCR studies showed that Tpo induces Tpo receptor (TpoR or c-mpl), whose expression was further potentiated by PDB addition. When the cells are induced with both PDB and Tpo erythropoietin receptor (EpoR) expression was inhibited. In the absence of Zn2+-induced PML/RARalpha expression, PDB and Tpo induced megakaryocytic differentiation of TF-1 MTPR clones as observed in 'wild-type' TF-1 cells. Conversely, when PML/RARalpha expression was induced by Zn2+, PDB and Tpo treatment of these clones caused only a reduced level of megakaryocytic differentiation. These observations indicate that: (1) TF-1 cells as well as other erythroleukemic cells, possess the capacity to differentiate to megakaryocytic cells when grown in the presence of protein kinase (PKC) activators and more efficiently when combined with Tpo; (2) the PML/RARalpha gene has a wide capacity to interfere with the program of hematopoietic differentiation, including megakaryocytic differentiation. Finally, we also observed that PML/RARalpha expression in TF-1 cells induces an up-modulation of interleukin-3 receptor, c-kit and c-mpl, a phenomenon which may offer these cells a growth advantage.

Since in vitro observations indicated that all-trans retinoic acid (ATRA), especially in combination with IFNalpha, can exert significant suppressive effects on Ph+ cells, we investigated the effects and the pharmacokinetic profile of ATRA in a selected cohort of patients with Ph+ chronic myeloid leukemia (CML) in chronic phase. Eighteen patients were treated with ATRA at a dose of 80 mg/m2/day (p.o.), divided into two equal doses after meals, for 7 consecutive days every other week for a maximum of 12 courses (1 course = 1 week on and 1 week off). Pharmacokinetic profiles of ATRA were evaluated during intermittent therapy on days 1 and 7 of course 1; on day 1 of course 2; on day 1 of course 6. Out of the 18 patients treated with ATRA, 11 (61%) went off study before the sixth course of treatment because of progressive hyperleukocytosis (seven cases), or thrombocytosis (one case), or refusal (three cases). Seven (39%) patients completed the first six courses (12 weeks) of treatment with ATRA and two of them (11%) maintained a white blood cell (WBC) <10 x 10[9]/l which was induced by the pretreatment with hydroxyurea. One patient completed the 12th course of ATRA maintaining WBC <10 x 10(9)/l, platelets <500 x 10(9)/l and spleen not palpable. The treatment with ATRA was well tolerated and only one patient discontinued the therapy because of non-hematological side-effects. The area under the concentration-time curve (AUC) decreased significantly (P< 0.001) during the first week of therapy. By adopting an intermittent dosing regimen, 1 week on/ 1 week off (1 course), at the start of courses 2 and 6, we obtained the ATRA AUCs equivalent to the ones achieved on day 1 of course 1. In conclusion, our results showed that ATRA alone appeared to be unable to control the WBC expansion in the CML patients in chronic phase. Moreover, it did not induce any remarkable cytoreductive effects on the platelet count and on the hemoglobin level. The major interest of ATRA would be in combination with other therapies. If ATRA was given in combination with IFNalpha or other agents, dose reduction of these would not be planned. On the basis of the pharmacokinetic profile, ATRA should be administered intermittently rather than continuously.

PML/RAR alpha is the putative transforming sequence of acute promyelocytic leukemias. We investigated the effects of PML/RAR alpha on cell survival by expressing the fusion protein in the growth factor-dependent TF-1 cell line and analyzing the kinetics of cell death after GM-CSF deprivation. Results showed that PML/RAR alpha expression markedly delayed apoptotic cell death (3 weeks vs 1 week) without inducing growth factor independence. Growth factor deprivation caused rapid and massive apoptosis of control TF-1 cells (>95% apoptotic cells after 4-5 days). Factor-deprived control cells were synchronously and irreversibly committed to apoptosis as shown by their inability to re-enter the cell cycle after GM-CSF re-addition. The percentage of apoptotic cells in the PML/RAR alpha expressing cells was low (approximately 30-35%) and constant over the 4 weeks of factor deprivation. GM-CSF re-addition produced rapid increase in cell number at all time points during the 4 weeks of factor deprivation, suggesting that commitment to apoptosis was asynchronous and delayed in PML/RAR alpha-expressing TF-1 cells. We conclude that PML/RAR alpha interferes with the genetic pathways which regulate survival by reducing the frequency of commitment to apoptosis. This biological effect of PML/RAR alpha may contribute to its leukemogenetic potential.

A rearrangement between the PML and RAR-alpha genes underlies the acute promyelocytic leukemia (APL)-specific t(15;17) translocation, leading to the production of a chimeric mRNA. Recent development of a reverse-transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha hybrid has proven useful for rapid diagnosis and monitoring of minimal residual disease (MRD) in APL patients. Preliminary studies in which the prognostic significance of RT-PCR was evaluated indicate that this test may identify patients at high risk of relapse.

Acute promyelocytic leukaemia is characterized by an expansion of haematopoietic precursors arrested at the promyelocytic stage (1). The differentiation block can be reversed by retinoic acid, which induces blast differentiation both in vitro (2) and in vivo (3-4). Acute promyelocytic leukaemia is also characterized by a 15;17 chromosome translocation (5) with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and within the PML gene, that encodes a putative transcription factor of unknown function (6-7), on 15 (8-10). As a consequence of the translocation a PML/RAR alpha gene is formed. It is transcriptionally active and encodes a PML/RAR alpha fusion protein detectable in all APL cases (11-14). We expressed the PML/RAR alpha protein in U937 myeloid precursor cell line and show that they: 1) lose the capacity to differentiate under the action of different stimuli (vitamin D3, transforming growth factor beta 1); ii) acquire enhanced sensitivity to retinoic acid; iii) exhibit a higher growth rate that is due to a reduction in apoptotic cell death. These results provide the first evidence of biological activity of PML/RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.

We performed clonality studies of hemopoietic reconstitution in 24 female patients (pts) with leukemias characterized by specific tumor markers. Thirteen pts had Acute Promyelocytic Leukemia (APL) with rearrangements of the RAR-a and PML genes, 8 BCR rearranged (BCR+)/Ph+ Chronic Myeloid Leukemia (CML) and 3 BCR+/Ph+ Acute Lymphoid Leukemia (ALL). Bone marrow (BM) DNA samples were obtained at diagnosis and at remission after Southern blot documented suppression of specific markers. The clonal or non-clonal nature of hemopoietic reconstitution was assessed by hybridizing the same DNAs with the M27 beta probe, in order to detect methylation differences at the X-linked DXS255 locus. Twenty four pts showed a polyclonal methylation pattern at remission, whereas in 3 cases an apparently clonal pattern was observed despite no evidence of specific gene rearrangement. In 2 of these 3 cases, however, DNAs derived from non-affected tissues (T lymphocytes, skin and BM fibroblasts) revealed the presence of the same DXS255 unmethylated allele detected at diagnosis, while in the third case we found the same apparently clonal pattern in blood mononuclear cells obtained from her healthy female BM donor. These data indicate that polyclonal hematopoiesis occur in APL and CML pts after therapy induced suppression of specific tumor markers, and that unbalanced or aberrant X chromosome methylation patterns are observed in some cases, most likely reflecting constitutional features.

A rearrangement between the PML and RAR-alpha genes underlies the acute promyelocytic leukemia (APL)-specific t(15;17) translocation, leading to the production of a chimeric mRNA. Recent development of a reverse-transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha hybrid has proven useful for rapid diagnosis and monitoring of minimal residual disease (MRD) in APL patients. Preliminary studies in which the prognostic significance of RT-PCR was evaluated indicate that this test may identify patients at high risk of relapse.