ALBERT

Description: The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 〉 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 〉 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.

Description: The aberrant function of transcription factors and/or kinase-based signaling pathways that regulate the ability of hematopoietic cells to proliferate, differentiate, and escape apoptosis accounts for the leukemic transformation of myeloid progenitors. Here, we demonstrate that simultaneous retinoid receptor ligation and blockade of the MEK/ERK signaling module, using the small-molecule inhibitor CI-1040, result in a strikingly synergistic induction of apoptosis in both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells with constitutive ERK activation. This proapoptotic synergism requires functional RAR and RXR retinoid receptors, as demonstrated using RAR- and RXR-selective ligands and RAR-defective cells. In the presence of MEK inhibitors, however, retinoid-induced chromatin remodeling, target-gene transcription, and granulocytic differentiation are strikingly inhibited and apoptosis induction becomes independent of death-inducing ligand/receptor pairs; this suggests that apoptosis induction by combined retinoids and MEK inhibitors is entirely distinct from the classical “postmaturation” apoptosis induced by retinoids alone. Finally, we identify disruption of Bcl-2–dependent mitochondrial homeostasis as a possible point of convergence for the proapoptotic synergism observed with retinoids and MEK inhibitors. Taken together, these results indicate that combined retinoid treatment and MEK blockade exert powerful antileukemic effects and could be developed into a novel therapeutic strategy for both AML and APL.

Description: The mammalian target of rapamycin (mTOR) pathway regulates major processes by integrating a variety of exogenous cues, including diverse environmental inputs in the tumor microenvironment (TME). In recent years, it has been well recognized that cancer cells co-exist and co-evolve with their TME, which is often involved in drug resistance. The mTOR pathway modulates the interactions between the stroma and the tumor, thereby affecting both the tumor immunity and angiogenesis. The activation of mTOR signaling is associated with these pro-oncogenic cellular processes, making mTOR a promising target for new combination therapies. This review highlights the role of mTOR signaling in the characterization and the activity of the TME’s elements and their implications in cancer immunotherapy.

Description: The MEK/ERK kinase module is frequently deregulated in human malignancies, including AML and MEL, and represents a promising therapeutic target. We have recently demonstrated that PD0325901, a selective MEK inhibitor, potently inhibits the growth of both AML and MEL cell lines (IC50 5–20 nM), through a combination of inhibition of cell cycle progression and induction of apoptosis. In order to identify functionally relevant targets of the activity of the MEK/ERK kinase module, we compared the gene expression profiles of the PD0325901-sensitive cell lines OCI-AML3 (AML, harboring a NPM gene mutation) and M14 (MEL, harboring a BRAF mutation), after exposure to vehicle control or 10 nM PD0325901 for 6 and 24h, using the Affymetrix U133A 2.0 and U133 Plus 2.0 gene chips, respectively. All analyses were conducted using the dChip software. In both models, the unsupervised approach showed a tight clustering that was both treatment- and time-dependent; unsupervised and supervised analyses highlighted that the greatest PD0325901-induced variations in gene expression were observed at 24h and 6h in the OCI-AML3 and M14 models, respectively. Comparison of the results obtained in the OCI-AML3 and M14 cell lines showed that 5 genes were consistently modulated after 6h, including CDC25A, cyclin G2, and DUSP6. Cyclin G2 and DUSP6 were also modulated after 24h, together with cyclin D1, TIMP3, and SEMA6A. On the contrary, MAFB, selected in both models, displayed a discordant behavior, being down-regulated upon treatment in OCI-AML3 and up-regulated in M14: this is consistent with the role of MAFB as an important regulator of hematopoiesis, while it does not appear to play a role in melanogenesis. Given the ability of PD0325901 to cause apoptosis, partly through induction of mitochondrial dysfunction in both AML and MEL models, we next investigated whether MEK inhibition specifically modulated the expression of genes related to mitochondrial homeostasis: also in this specific context, the effects were more rapidly induced in M14 than in OCI-AML3, with the greatest changes observed within 6h in the M14 cell line. Two genes were down-regulated after 24h in both models: PPIF, supposedly involved in apoptosis induction, and GRPEL1, on which little is currently known. In addition to mitochondrion-specific changes in gene expression, expression of the mitochondrial protein CPT1A significantly decreased (2-fold), as assessed by WB, after 6 h of exposure of OCI-AML3 cells to 10 nM PD0325901, under which conditions ß-oxidation was also suppressed by the drug. Overall, these results indicate that MEK inhibition by PD0325901 results in the modulation of the expression of a small number of genes that are common to ontogenetically distant tumor models, thereby representing attractive candidates for a shared, functionally relevant, MAPK activation signature; moreover, MEK inhibition results in profound intracellular metabolic changes that might importantly contribute to the anti-neoplastic effect of PD0325901 in AML and, possibly, in other tumor models. MM and SC equally contributed to the work presented.

Description: Abstract 594 Our group has recently reported on the growth inhibitory and pro-apoptotic effects of the MEK inhibitor PD0325901 in preclinical models of hematologic malignancies, particularly AML. However, the molecular mechanisms of AML sensitivity/resistance to MEK inhibitors remain elusive. Regardless of their sensitivity to PD0325901, none of the AML, ALL, and multiple myeloma cell lines examined harbored HRAS, BRAF, MEK, and PI3K mutations, while NB4, KMS18, and CEM had a mutated KRAS. In the absence of unequivocal genetic predictors of sensitivity/resistance to MEK inhibitors, one possibile alternative was to pursue rational, mechanism-based combinations with agents interfering with putative ‘escape' pathways. Therefore, we analyzed signaling along the ERK and AKT pathways in three different models of PD0325901 resistance: intrinsically resistant cell lines (U937), sensitive cell lines (OCI-AML3) that had been rendered resistant by prolonged exposure to another MEK inhibitor (CI-1040), and cytokine-exposed, non-responding FDC-P1 versus responding, v-fms-transformed FDC-P1 cells. Resistant U937 had low basal levels of phosphorylated ERK that were not completely abrogated by PD0325901 treatment even at high (1000 nM) concentrations; similarly, at least 100-fold higher PD0325901 concentrations were required to abrogate ERK phosphorylation in resistant IL-3-cultured FDC-P1 and OCI-AML3, as compared with their sensitive counterparts. Moreover, in the OCI-AML3 model, MEK inhibition-induced growth inhibition directly paralleled the ability of either PD0325901 or CI-1040 to abrogate ERK phosphorylation. Interestingly, PD0325901 induced AKT phosphorylation (S473) in all three models of resistance; phosphoproteomic analysis confirmed increased signaling through the PI3K/AKT/mTOR pathway upon MEK inhibition (10 nM PD0325901), with increased AKT1 (S473), mTOR (S2448), and S6Ka (T389) phosphorylation and increased PI3K expression. In OCI-AML3 resistance to MEK blockade-mediated growth inhibition clearly correlated with higher levels of AKT phosphorylation. Notably, comparative proteomic analysis of PD0325901-sensitive (OCI-AML3) and -resistant (U937) AML cell lines revealed upregulation of PI3K expression (+62%), increased AKT1 expression (+53%) and phosphorylation (T308, +86%; S473, +53%), inhibitory PTEN phosphorylation (S380+S382+S385, +46%), increased S6Ka and b expression (+48%), and increased S6 phosphorylation (S235, +54%) in U937 cells. From a functional standpoint, combined MEK inhibition (PD0325901) and mTOR blockade downstream of AKT (Temsirolimus) resulted in a striking growth-inhibitory synergism in OCI-AML3, with an average combination index at the ED50, ED75, and ED90 of 0.3±0.2. Similar results were obtained in the Flt3/ITD cell line MOLM-13. Overall, these results suggest that resistance to PD0325901-mediated growth inhibition may stem from a combination of decreased efficiency towards ERK inhibition and increased parallel signaling through AKT. The latter is an emerging theme in cancer cell signal transduction, as highlighted by recent reports of a functional cross-talk between the MEK/ERK and PI3K/AKT/mTOR in different models of cancer progression and response to individual pathway inhibitors. These findings bear potentially important consequences for clinical translation, as we show that combined blockade of both MEK and mTOR signaling, a therapeutic strategy that has shown promise in different solid tumor models, results in a strongly synergistic inhibition of leukemic cell growth. Disclosures: No relevant conflicts of interest to declare.

Description: In hematologic malignancies, constitutive activation of the Raf/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Indeed, we have recently demonstrated that selective MEK-I potently inhibit the growth of AML cell lines and ex vivo-cultured primary AML blasts (Blood2006, 108:254). However, these effects are mostly related to the inhibition of cell cycle progression, while apoptosis induction requires higher concentrations of the inhibitors and longer times of exposure. Thus, we investigated MEK-I-induced changes in phospho-protein expression and gene expression profiles, in order to identify relevant downstream targets and to design rational MEK-I-based combination strategies. Analysis of phosphorylation levels of 18 different target proteins performed in OCI-AML3 cells indicated that MEK blockade induces, among other effects, an over-activation of RAF and MEK, suggesting the interruption of a negative feedback loop. Moreover, gene expression profiling indicated that, in the same cellular model, MEK-I induced upregulation of the Flt-3 receptor. Based on these observations, as well as on recent evidence indicating that the Raf inhibitor sorafenib directly inhibits signaling through Flt-3 (JNCI2008, 100:184), experiments were performed in OCI-AML3 and MOLM-13 (which harbors a Flt3 ITD) cells to test the activity of MEK-I in combination with sorafenib. Simultaneous inhibition of Flt3/Raf and MEK resulted in the synergistic inhibition of cell growth, as measured by isobologram analysis (Chou–Talalay method) in both model systems, with combination indexes (CI) of 0.12 and 0.48 for OCI-AML3 and MOLM-13 cells, respectively. Neither sorafenib nor MEK-I induced apoptosis in either cell line when used alone; however, apoptosis was observed in up to 50% of the cells with the combined treatment. Based on our previous experience, as well as on the ability of MEK-I to modulate the expression, among others, of genes controlling mitochondrial homeostasis (e.g. PPIF, GRPEL1), we next investigated the impact of simultaneous inhibition of the MEK and Bcl-2 pathways in AML cells. Exposure of OCI-AML3 and MOLM-13 cells to a combination of MEK-I and the Bcl-2/Bcl-xL inhibitor, ABT-737 (kindly provided by Abbott Laboratories) synergistically inhibited cell growth, with CI ranging from 0.45 to 0.04 in OCI-AML3 and from 0.75 to 0.14 in MOLM-13, respectively. In both cellular models, ABT-737dose-dependently induced apoptosis, while MEK-I, at the concentrations used in combination experiments, did not appreciably increase apoptotic cell death; however, simultaneous Bcl- 2/Bcl-xL inhibition and MEK blockade resulted in the massive induction of apoptosis (up to 85% and 67% net apoptosis induction in OCI-AML3 and MOLM-13 cells, respectively). Such pro-apoptotic interaction was highly synergistic with CI of 0.18 and 0.16 in OCIAML3 and MOLM-13 cells, respectively. In contrast, combination with MEK-I did not appreciably sensitize the MEK-I-resistant cell line U937 to either sorafenib- or ABT-737- induced growth inhibitory and pro-apoptotic effects. Overall these results support the role of the Raf/MEK/ERK kinase module as a prime target for the molecular therapy of AML and suggest that both “vertical” and “lateral” combination strategies based on MEK inhibition may produce highly synergistic anti-leukemic effects.

Description: In the MAPK module, MEK lies upstream of ERK which is found constitutively activated in a host of human tumors. We already demonstrated the growth inhibitory activity of MEK inhibitors in myeloid cells (JCI 2001, Leukemia 2005). PD0325901 is the latest small-molecule inhibitor of MEK with promising effects at lower concentrations. We tested its activity (0.1–1000 nM) in a broad spectrum of leukemia, melanoma and breast cancer cell lines evaluating changes on cell cycle distribution, apoptosis, protein and gene expression profiles, aiming at defining the molecular signature induced by this MEK inhibitor. Among hematopoietic cell lines, PD0325901 induced a marked growth inhibition in myeloid cells with constitutive ERK activation (IC50=11 and 12 nM for OCI-AML3 and OCI-AML2, respectively). Conversely, relative resistance to PD0325901-mediated growth inhibition (IC50〉1μM) was observed in myeloid cell lines without constitutive ERK activation (U937, KG-1) and in lymphoid cell lines (Raji, Jurkat). Among the solid tumor cell lines, the M14 melanoma was markedly sensitive to PD0325901-induced growth inhibition (IC50=24 nM), even with forced Bcl-2 expression (IC50 ranging from 64 to 200 nM in Bcl-2-overexpressing clones). Conversely, the breast cancer cell lines tested (SKBr3, BT474, MDA-MB-231 and ZR75-1) proved relatively resistant (IC50≥1 μM), regardless of the ERK phosphorylation status. In responsive cells (OCI-AML3, OCI-AML2 and M14), PD0325901 inhibited ERK phosphorylation in a dose-dependent manner, the effect was already evident at 15 min. Cell cycle distribution analysis demonstrated a dramatic dose-dependent decrease in the proliferative compartment in OCI-AML3. Subsequently (48–72 h), PD0325901 induced apoptosis in a dose- and time-dependent fashion, as demonstrated by the increase in the percentage of AnnV+ cells from 6.3%±1.1 (DMSO control) to 15.5%±3.9, 31.5%±2.8 and 45.3%±0.14 (10, 100, and 1000 nM PD0325901, respectively). Gene expression profiling was conducted using AffymetrixTM HG-U133A 2.0 GeneChip® in OCI-AML3 cells exposed to DMSO (control) or PD0325901 at 10nM. Using a p

Description: Mounting preclinical and clinical evidence indicates that rewiring the host immune system in favor of an antitumor microenvironment achieves remarkable clinical efficacy in the treatment of many hematological and solid cancer patients. Nevertheless, despite the promising development of many new and interesting therapeutic strategies, many of these still fail from a clinical point of view, probably due to the lack of prognostic and predictive biomarkers. In that respect, several data shed new light on the role of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN) in affecting the composition and function of the tumor microenvironment (TME) as well as resistance/sensitivity to immunotherapy. In this review, we summarize current knowledge on PTEN functions in different TME compartments (immune and stromal cells) and how they can modulate sensitivity/resistance to different immunological manipulations and ultimately influence clinical response to cancer immunotherapy.

Description: The threatening notoriety of pancreatic cancer mainly arises from its negligible early diagnosis, highly aggressive progression, failure of conventional therapeutic options and consequent very poor prognosis. The most important driver genes of pancreatic cancer are the oncogene KRAS and the tumor suppressors TP53, CDKN2A, and SMAD4. Although the presence of few drivers, several signaling pathways are involved in the oncogenesis of this cancer type, some of them with promising targets for precision oncology. Pancreatic cancer is recognized as one of immunosuppressive phenotype cancer: it is characterized by a fibrotic-desmoplastic stroma, in which there is an intensive cross-talk between several cellular (e.g., fibroblasts, myeloid cells, lymphocytes, endothelial, and myeloid cells) and acellular (collagen, fibronectin, and soluble factors) components. In this review; we aim to describe the current knowledge of the genetic/biological landscape of pancreatic cancer and the composition of its tumor microenvironment; in order to better direct in the intrinsic labyrinth of this complex tumor type. Indeed; disentangling the genetic and molecular characteristics of cancer cells and the environment in which they evolve may represent the crucial step towards more effective therapeutic strategies

Description: Inflammation might substantially contribute to the limited therapeutic success of current systemic therapies in colorectal cancer (CRC). Amongst cytokines involved in CRC biology, the proinflammatory chemokine IL-8 has recently emerged as a potential prognostic/predictive biomarker. Here, we show that BRAF mutations and PTEN-loss are associated with high IL-8 levels in CRC models in vitro and that BRAF/MEK/ERK, but not PI3K/mTOR, targeting controls its production in different genetic contexts. In particular, we identified a BRAF/ERK2/CHOP axis affecting IL-8 transcription, through regulation of CHOP subcellular localization, and response to targeted inhibitors. Moreover, RNA Pol II and an open chromatin status in the CHOP-binding region of the IL-8 gene promoter cooperate towards increased IL-8 expression, after a selective BRAF inhibition. Overall, our data show that IL-8 production is finely and differentially regulated depending on the tumor genetic context and might be targeted for therapeutic purposes in molecularly defined subgroups of CRC patients.