Eduardo M. Rego

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Eμ-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Eμ-Myc/+ mice. When accurate translational control is re-established in Eμ-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

Two Critical Hits for Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations that always involve the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes). Due to the reciprocity of the translocation, X-RARalpha and RARalpha-X fusion proteins coexist in APL blasts. PLZF-RARalpha transgenic mice (TM) develop leukemia that lacks the differentiation block at the promyelocytic stage that characterizes APL. We generated TM expressing RARalpha-PLZF and PLZF-RARalpha in their promyelocytes. RARalpha-PLZF TM do not develop leukemia. However, PLZF-RARalpha/RARalpha-PLZF double TM develop leukemia with classic APL features. We demonstrate that RARalpha-PLZF can interfere with PLZF transcriptional repression and that this is critical for APL pathogenesis, since leukemias in PLZF(-/-)/PLZF-RARalpha mutants and in PLZF-RARalpha/RARalpha-PLZF TM are indistinguishable. Thus, both products of a cancer-associated translocation are crucial in determining the distinctive features of the disease.

Active Pin1 is a key target of all- trans retinoic acid in acute promyelocytic leukemia and breast cancer

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)—a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive—inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML–RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.

Leukemia with distinct phenotypes in transgenic mice expressing PML/RAR alpha, PLZF/RAR alpha or NPM/RAR alpha.

Recurrent chromosomal translocations involving the RARα locus on chromosome 17 are the hallmark of acute promyelocytic leukemia (APL). The RARα gene fuses to variable partners (PML, PLZF, NPM, NuMA and STAT5B: X genes) leading to the expression of APL-specific fusion proteins with identical RARα moieties. To analyse whether the variable X moiety could affect the activity of the fusion protein in vivo, we generated and characterized, on a comparative basis, NPM/RARα transgenic mice (TM) in which the fusion gene is expressed under the control of a human Cathepsin G (hCG) minigene. We compared the features of the leukemia observed in these TM with those in hCG-PML/RARα and hCG-PLZF/RARα TM. In all three transgenic models, leukemia developed after a variably long latency, with variable penetrance. However, the three leukemias displayed distinct cytomorphological features. hCG-NPM/RARα leukemic cells resembled monoblasts. This phenotype contrasts with what was observed in the hCG-PML/RARα TM model in which the leukemic phase was characterized by the proliferation of promyelocytic blasts. Similarly, hCG-PLZF/RARα TM displayed a different phenotype where terminally differentiated myeloid cells predominated. Importantly, the NPM/RARα oncoprotein was found to localize in the nucleolus, unlike PML/RARα and PLZF/RARα, thus possibly interfering with the normal function of NPM. Similarly to what was observed in human APL patients, we found that NPM/RARα and PML/RARα, but not PLZF/RARα leukemia, was responsive to all-trans retinoic acid (ATRA) or As2O3 treatments. Taken together, our results underscore the critical relevance of the X moiety in dictating the biology of the disease and the activity of the APL fusion oncoprotein.

Determination of P‐glycoprotein, MDR‐related protein 1, breast cancer resistance protein, and lung‐resistance protein expression in leukemic stem cells of acute myeloid leukemia

The most primitive leukemic precursor in acute myeloid leukemia (AML) is thought to be the leukemic stem cell (LSC), which retains the properties of self‐renewal and high proliferative capacity and quiescence of the hematopoietic stem cell. LSC seems to be immunophenotypically distinct and more resistant to chemotherapy than the more committed blasts. Considering that the multidrug resistance (MDR) constitutive expression may be a barrier to therapy in AML, we have investigated whether various MDR transporters were differentially expressed at the protein level by different leukemic subsets.

Improving acute promyelocytic leukemia (APL) outcome in developing countries through networking, results of the International Consortium on APL.

Thanks to modern treatment with all-trans retinoic acid and chemotherapy, acute promyelocytic leukemia (APL) is now the most curable type of leukemia. However, this progress has not yielded equivalent benefit in developing countries. The International Consortium on Acute Promyelocytic Leukemia (IC-APL) was established to create a network of institutions in developing countries that would exchange experience and data and receive support from well-established US and European cooperative groups. The IC-APL formulated expeditious diagnostic, treatment, and supportive guidelines that were adapted to local circumstances. APL was chosen as a model disease because of the potential impact on improved diagnosis and treatment. The project included 4 national coordinators and reference laboratories, common clinical record forms, 5 subcommittees, and laboratory and data management training programs. In addition, participating institutions held regular virtual and face-to-face meetings. Complete hematological remission was achieved in 153/180 (85%) patients and 27 (15%) died during induction. After a median follow-up of 28 months, the 2-year cumulative incidence of relapse, overall survival (OS), and disease-free survival (DFS) were 4.5%, 80%, and 91%, respectively. The establishment of the IC-APL network resulted in a decrease of almost 50% in early mortality and an improvement in OS of almost 30% compared with historical controls, resulting in OS and DFS similar to those reported in developed countries.

Thalidomide plus dexamethasone as a maintenance therapy after autologous hematopoietic stem cell transplantation improves progression-free survival in multiple myeloma

Despite the good response of stem cell transplant (SCT) in the treatment of multiple myeloma (MM), most patients relapse or do not achieve complete remission, suggesting that additional treatment is needed. We assessed the impact of thalidomide in maintenance after SCT in untreated patients with MM. A hundred and eight patients (<70 years old) were randomized to receive maintenance with dexamethasone (arm A; n = 52) or dexamethasone with thalidomide (arm B; n = 56; 200 mg daily) for 12 months or until disease progression. After a median follow‐up of 27 months, an intention to treat analysis showed a 2‐year progression‐free survival (PFS) of 30% in arm A (95% CI 22–38) and 64% in arm B (95% CI 57–71; P = 0.002), with median PFS of 19 months and 36 months, respectively. In patients who did not achieve at least a very good partial response, the PFS at 2 years was significantly higher when in use of thalidomide (19 vs. 59%; P = 0.002). Overall survival at 2 years was not significantly improved (70 vs. 85% in arm A and arm B, respectively; P = 0.27). The addition of thalidomide to dexamethasone as maintenance improved the PFS mainly in patients who did not respond to treatment after SCT. Am. J. Hematol.

Reciprocal products of chromosomal translocations in human cancer pathogenesis: key players or innocent bystanders?

Chromosomal translocations are frequently involved in the pathogenesis of leukemias, lymphomas and sarcomas. They can lead to aberrant expression of oncogenes or the generation of chimeric proteins. Classically, one of the products is thought to be oncogenic. For example, in acute promyelocytic leukaemia (APL), reciprocal chromosomal translocations involving the retinoic acid receptor alpha (RARalpha) gene lead to the formation of two fusion genes: X-RARalpha and RARalpha-X (where X is the alternative RARalpha fusion partner: PML, PLZF, NPM, NuMA and STAT 5b). The X-RARalpha fusion protein is indeed oncogenic. However, recent data indicate that the RARalpha-X product is also critical in determining the biological features of this leukemia. Here, we review the current knowledge on the role of reciprocal products in cancer pathogenesis, and highlight how their expression might impact on the biology of their respective tumour types.

Adhesion molecules and differentiation syndrome: phenotypic and functional analysis of the effect of ATRA, As2O3, phenylbutyrate, and G-CSF in acute promyelocytic leukemia

Background and Objectives Differentiation Syndrome (DS) is a treatment complication which can occur in patients treated with acute promyelocytic leukemia (APL) with all transretinoic acid (ATRA) or As2O3, and is characterized by enhanced leukocyte transmigration. As2O3, Phenylbutyrate (PB) and G-CSF are known to potentiate ATRA effects. Our aim was to analyze the changes in expression and function of adhesion molecules induced by ATRA, As2O3, G-CSF and PB, and their association. Design and Methods APL blasts and NB4 cells were treated with ATRA, As2O3, PB, G-CSF or their association and the expression of adhesion molecules was determined by flow cytometry. Cell adhesion was evaluated in vitro using Matrigel and for the in vivo analysis, Balb-c mice were injected with NB4 cells pre-treated with ATRA, As2O3, ATRA+G-CSF or ATRA+As2O3. In addition, CD54 and CD18 knock-out mice were injected with NB4 cells and concomitantly treated with ATRA. In both models, the MPO activity in the lungs was determined 6 hours after the injection of the cells. Results In NB4 and APL blasts, ATRA and As2O3 increased CD54 expression, but no synergism was detected. CD11b and CD18 were also up-regulated by ATRA in primary cells. PB and G-CSF had no effect, but the latter potentiated ATRA-induced CD18 up-regulation. These changes were accompanied by increased adhesion to Matrigel and to lung microvasculature, and reversed by anti-CD54, anti-CD18 antibodies. In CD54 and CD18 knock-out mice the ATRA effect was canceled. Interpretation and Conclusions The use of As2O3, PB and G-CSF in association with ATRA should not aggravate DS in APL.

Antinociception induced by intraperitoneal injection of gentamicin in rats and mice

&NA; Intraperitoneal administration of gentamicin sulfate (5–800 &mgr;g/kg), but not gentamicin base (23–92 &mgr;g/kg) produced antinociception in rats and mice, as assessed by the tail‐flick, carrageenan‐induced articular incapacity tests, and hot‐plate tests. The AD50 s in rats (tail‐flick test) and mice (hot‐plate test) were 11.48 and 147.9 &mgr;g/kg, respectively, but doses of 200–800 &mgr;g/kg were required to reduce the hyperalgesia induced in rats by carrageenan. In both species, bell‐shaped dose‐response curves were obtained, indicating that high doses of gentamicin had little or no effect. Non‐effective doses of gentamicin failed to produce a significant increase in morphine antinociception in either rodent species. The possible involvement of N‐type voltage‐sensitive. Ca2+ channels in the mechanism of antinociception induced by gentamicin is considered.