Manuela Mancini

Identification of the first non-peptidic small molecule inhibitor of the c-Abl/14-3-3 protein–protein interactions able to drive sensitive and Imatinib-resistant leukemia cells to apoptosis

An in silico structure-based ligand design approach resulted in the identification of the first non-peptidic small molecule able to inhibit protein-protein interactions between 14-3-3 and c-Abl. This compound shows an anti-proliferative effect on human leukemia cells either sensitive or resistant to Imatinib, in consequence of the T315I mutation. It also mediates c-Abl release from 14-3-3 in a way similar to that found in response to Imatinib treatment.

RAD 001 (everolimus) prevents mTOR and Akt late re-activation in response to imatinib in chronic myeloid leukemia

The mammalian target of rapamycin (mTOR) is one target of BCR‐ABL fusion gene of chronic myeloid leukemia (CML). Moreover, it drives a compensatory route to Imatinib mesylate (IM) possibly involved in the progression of leukemic progenitors towards a drug‐resistant phenotype. Accordingly, mTOR inhibitors are proposed for combined therapeutic strategies in CML. The major caveat in the use of mTOR inhibitors for cancer therapy comes from the induction of an mTOR‐phosphatidylinositol 3 kinase (PI3k) feedback loop driving the retrograde activation of Akt. Here we show that the rapamycin derivative RAD 001 (everolimus, Novartis Institutes for Biomedical Research) inhibits mTOR and, more importantly, revokes mTOR late re‐activation in response to IM. RAD 001 interferes with the assembly of both mTOR complexes: mTORC1 and mTORC2. The inhibition of mTORC2 results in the de‐phosphorylation of Akt at Ser473 in the hydrophobic motif of C‐terminal tail required for Akt full activation and precludes Akt re‐phosphorylation in response to IM. Moreover, RAD 001‐induced inhibition of Akt causes the de‐phosphorylation of tuberous sclerosis tumor suppressor protein TSC2 at 14‐3‐3 binding sites, TSC2 release from 14‐3‐3 sigma (restoring its inhibitory function on mTORC1) and nuclear import (promoting the nuclear translocation of cyclin‐dependent kinase [CDK] inhibitor p27Kip1, the stabilization of p27Kip1 ligand with CDK2, and the G0/G1 arrest). RAD 001 cytotoxicity on cells not expressing the BCR‐ABL fusion gene or its p210 protein tyrosine kinase (TK) activity suggests that the inhibition of normal hematopoiesis may represent a drug side effect. J. Cell. Biochem. 109: 320–328, 2010.

A New Nonpeptidic Inhibitor of 14-3-3 Induces Apoptotic Cell Death in Chronic Myeloid Leukemia Sensitive or Resistant to Imatinib

Resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitor imatinib mesylate (IM) is most often due to point mutations in the Bcr-Abl fusion gene. T315I mutation (resulting in substitution of Ile for a Thr residue at the “gatekeeper” position 315) raises particular concern, because it also provides resistance to second-generation kinase inhibitors already approved for clinical use (nilotinib and dasatinib). Much effort is therefore focused on alternative molecular-based strategies. Previous studies proved that binding to 14-3-3 scaffolding proteins leads to cytoplasmic compartmentalization and suppression of proapoptotic and antiproliferative signals associated with Bcr-Abl protein kinase, hence contributing to leukemic clone expansion. Here we investigated the effect of 14-3-3 inhibition disruption on hematopoietic cells expressing the IM-sensitive wild type Bcr-Abl and the IM-resistant T315I mutation. Using a virtual screening protocol and docking simulations, we identified a nonpeptidic inhibitor of 14-3-3, named BV02, that exhibits a remarkable cytotoxicity against both cell types. c-Abl release from 14-3-3σ, promoting its relocation to nuclear compartment (where it triggers transcription of p73-dependent proapoptotic genes) and to mitochondrial membranes (where it induces the loss of mitochondrial transmembrane potential) combined with c-Abl enhanced association with caspase 9 (a critical step of sequential caspase activation further contributing to c-Abl pro-apoptotic function) has a prominent role in the effect of BV02 on Bcr-Abl-expressing cells. In conclusion, BV02 may be considered as a treatment option for CML and, in particular, for more advanced phases of the disease that developed IM resistance as a consequence of Bcr-Abl point mutations.

Zinc-finger transcription factor slug contributes to the survival advantage of chronic myeloid leukemia cells

Slug, a Snail-related zinc-finger transcription factor implicated in the increased motility of mesenchymal cells during embryonic development and progression of cancer cells towards an invasive phenotype, plays a specific and critical role in the pathogenesis of Bcr-Abl-associated leukemias. Here we report that Slug over-expression associated with Bcr-Abl is conditional upon the tyrosine kinase (TK) activity of 210 fusion protein. Slug over-expression is driven by transcriptional events eventually integrated by post-transcriptional mechanisms leading to protein stabilization and is at least partly regulated by the ERK1/2 mitogen-activated protein kinase (MAPK). It contributes to apoptosis resistance of leukemic progenitors through the repression of pro-apoptotic Puma. Moreover, Slug is a component of leukemic progenitor resistance to imatinib mesylate (IM) driven by Bcr-Abl point mutations and, in particular, by T315I. Slug over-expression associated with p210 Bcr-Abl TK either in the wild type (wt) or mutated conformation results in a significant reduction of E-cadherin, the substrate of Beta catenin at cell membranes. In conclusion, our results suggest that Slug has a central role in a complex network involved in prolonged survival and IM resistance of CML progenitors.

Mutations in the BCR-ABL1 Kinase Domain and Elsewhere in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) has been the first human malignancy to be associated, more than 50 years ago, with a consistent chromosomal abnormality--the t(9;22)(q34;q11) chromosomal translocation. The resulting BCR-ABL1 fusion gene, encoding a tyrosine kinase with deregulated activity, has a central role in the pathogenesis of CML. Ancestral or additional genetic events necessary for CML to develop have long been hypothesized but never really demonstrated. CML can successfully be treated with tyrosine kinase inhibitors (TKIs). Mutations in the BCR-ABL1 kinase domain might arise, however, that confer resistance to 1 or more of the currently available TKIs. Hence, the critical role of BCR-ABL1 mutation screening for optimal therapeutic management, with the current gold standard technique, conventional sequencing, likely to be replaced soon by ultra-deep sequencing. Mutations in genes other than BCR-ABL1 include ASXL1, TET2, RUNX1, DNMT3A, EZH2, and TP53 in chronic phase patients and RUNX1, ASXL1, IKZF1, WT1, TET2, NPM1, IDH1, IDH2, NRAS, KRAS, CBL, TP53, CDKN2A, RB1, and GATA-2 mutations in advanced phase patients. The latter also display additional cytogenetic abnormalities, including submicroscopic regions of gain or loss that only single nucleotide polymorphism arrays or array comparative genomic hybridization can detect. Whether whole genome/exome sequencing studies will uncover novel mutations relevant for pathogenesis, progression, and risk-adapted therapy is still unclear.

mTOR inhibitor RAD001 (Everolimus) enhances the effects of imatinib in chronic myeloid leukemia by raising the nuclear expression of c-ABL protein

Constitutive tyrosine kinase (TK) activity of p210 BCR-ABL fusion protein of chronic myeloid leukemia (CML) usurps physiological functions of normal p145 c-ABL protein. Accordingly, its inhibition by imatinib mesylate (IM) lets p145 c-ABL translocate into the nuclear compartment, which drives cell growth arrest and apoptotic death. Here we show that IM and the mammalian target of rapamycin (mTOR) inhibitor RAD001 (Everolimus) have additive effects on BCR-ABL-expressing cells. Those effects are at least partly conditional upon the enhanced nuclear accumulation of p145 c-ABL through events encompassing post-translational modifications of p145 c-ABL (Thr(735) phosphorylation) precluding its nuclear export and of 14-3-3 sigma (Ser(186) phosphorylation by c-Jun N-terminal kinase [JNK]) promoting p145 c-ABL nuclear re-import.

Computational techniques are valuable tools for the discovery of protein–protein interaction inhibitors: The 14-3-3σ case

Targeting the binding site of 14-3-3 proteins lets the release of partner proteins involved in cell cycle progression, apoptosis, cytoskeletal rearrangement and transcriptional regulation and may therefore be regarded as an alternative strategy to integrate conventional therapeutic approaches against cancer. In the present work, we report the identification of two new small molecule inhibitors of 14-3-3σ/c-Abl protein-protein interaction (BV01 and BV101) discovered by means of computational methods. The most interesting compound (BV01) showed a lethal dose (LD(50)) in the low micromolar range against Ba/F3 murine cell lines expressing the Imatinib (IM)-sensitive wild type Bcr-Abl construct and the IM-resistant Bcr-Abl mutation T315I. BV01 interaction with 14-3-3σ was demonstrated by NMR studies and elucidated by docking. It blocked the binding domain of 14-3-3σ, hence promoting the release of the partner protein c-Abl (the one not involved in Bcr rearrangement), and its translocation to both the nuclear compartment and mitochondrial membranes to induce a pro-apoptotic response. Our results advance BV01 as a confirmed hit compound capable of eliciting apoptotic death of Bcr-Abl-expressing cells by interfering with 14-3-3σ/c-Abl protein-protein interaction.

Endoplasmic reticulum stress initiates apoptotic death induced by STI571 inhibition of p210 bcr-abl tyrosine kinase

The endoplasmic reticulum (ER) is the site where proteins destined to either secretion or different subcellular compartments assemble and the major storage of intracellular Ca(2+). The ER stress resulting from a variety of toxic insults leads to apoptosis. Here, we showed that the apoptotic death triggered by STI571, an inhibitor of the p210 bcr-abl tyrosine kinase, in murine myeloid progenitors transducing the p210 bcr-abl tyrosine kinase of Chronic Myeloid Leukemia (CML) proceeds from ER stress. The Bcl-2 dowmodulation and inactivation induced by the binding to its antagonist: Bad, the release of caspase 12 from the ER membranes in its active form and of Ca(2+) from the ER pool addressed towards ER a sensor of STI571-induced pro-apoptotic signal.

Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44

The processivity factor of human cytomegalovirus DNA polymerase, phosphoprotein ppUL44, is essential for viral replication. During viral infection ppUL44 is phosphorylated by the viral kinase pUL97, but neither the target residues on ppUL44 nor the effect of phosphorylation on ppUL44s activity are known. We report here that ppUL44 is phosphorylated when transiently expressed in mammalian cells and coimmunoprecipitates with cellular kinases. Of three potential phosphorylation sites (S413, S415, S418) located upstream of ppUL44s nuclear localization signal (NLS) and one (T427) within the NLS itself, protein kinase CK2 (CK2) specifically phosphorylates S413, to trigger a cascade of phosphorylation of S418 and S415 by CK1 and CK2, respectively. Negative charge at the CK2/CK1 target serine residues facilitates optimal nuclear accumulation of ppUL44, whereas negative charge on T427, a potential cyclin-dependent 1 phosphorylation site, strongly decreases nuclear accumulation. Thus, nuclear transport of ppUL44 is finely tuned during viral infection through complex phosphorylation events.

14‐3‐3 Ligand Prevents Nuclear Import of c‐ABL Protein in Chronic Myeloid Leukemia

Here we demonstrated that the ‘loss of function’ of not‐rearranged c‐ABL in chronic myeloid leukemia (CML) is promoted by its cytoplasmic compartmentalization bound to 14‐3‐3 sigma scaffolding protein. In particular, constitutive tyrosine kinase (TK) activity of p210 BCR‐ABL blocks c‐Jun N‐terminal kinase (JNK) phosphorylation leading to 14‐3‐3 sigma phosphorylation at a critical residue (Ser186) for c‐ABL binding in response to DNA damage. Moreover, it is associated with 14‐3‐3 sigma over‐expression arising from epigenetic mechanisms (promoter hyper‐acetylation). Accordingly, p210 BCR‐ABL TK inhibition by the TK inhibitor Imatinib mesylate (IM) evokes multiple events, including JNK phosphorylation at Thr183, p38 mitogen‐activated protein kinase (MAPK) phosphorylation at Thr180, c‐ABL de‐phosphorylation at Ser residues involved in 14‐3‐3 binding and reduction of 14‐3‐3 sigma expression, that let c‐ABL release from 14‐3‐3 sigma and nuclear import, and address BCR‐ABL‐expressing cells towards apoptotic death. Informational spectrum method (ISM), a virtual spectroscopy method for analysis of protein interactions based on their structure, and mathematical filtering in cross spectrum (CS) analysis identified 14‐3‐3 sigma/c‐ABL binding sites. Further investigation on CS profiles of c‐ABL‐ and p210 BCR‐ABL‐containing complexes revealed the mechanism likely involved 14‐3‐3 precluded phosphorylation in CML cells.