PRECLINICAL RATIONALE FOR THE USE OF THE AKT INHIBITOR PERIFOSINE IN COMBINATION WITH THE MULTIKINASE INHIBITOR SORAFENIB IN HODGKIN LYMPHOMA

INTRODUCTION: A significant proportion of Hodgkin lymphoma (HL) patients refractory to first-line chemotherapy or relapsing after autologous transplantation are not cured with currently available treatments and require new treatments. The PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL. These pathways can be targeted using the AKT inhibitor perifosine (\uc6terna Zentaris GmBH, Germany, EU), and the RAF/MEK/ERK inhibitor sorafenib (Nexavar\uae, Bayer, Germany, EU). We hypothesized that perifosine in combination with sorafenib might have a therapeutic activity in HL by overcoming the cytoprotective and anti-apoptotic effects of PI3K/Akt and RAF/MEK/ERK pathways. Since preclinical evidence supporting the anti-lymphoma effects of the perifosine/sorafenib combination are still lacking, the present study aimed at investigating in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice using tumor growth rates and survival as endpoints. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P 64.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of apoptosis. In responsive cell lines, WB analysis showed that anti-proliferative events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P 64.0001) as well as mice receiving perifosine alone (49 days, P 64.03) or sorafenib alone (54 days, P 64.007). In addition, perifosine/sorafenib treatment had no effect on HDLM-2 nodules, but significantly reduced L-540 nodules with 50% tumor growth inhibition (P 64.0001), compared to controls. In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P 64.0001) and necrosis (2- to 8-fold, P 64.0001), as compared to controls or treatment with single agents. CONCLUSIONS: Perifosine/sorafenib combination resulted in potent anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation in HL patients

CDK5 PLAYS A KEY ROLE IN PROLIFERATION, APOPTOSIS, AND IN VIVO TUMOR GROWTH OF DIFFUSE LARGE B-CELL LYMPHOMA

Diffuse Large B-cell lymphoma (DLBCL) is the most common adult non-Hodgkin lymphoma, whose standard of care is the immunochemotherapy R-CHOP. Chemorefractory patients, still approximately 40%, represent an unmet medical need, requiring novel targets for innovative treatments. One potential target is cyclin-dependent kinase 5 (CDK5), a serine/threonine protein kinase that has been recently linked to tumor development and progression. Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine protein kinase, which forms active complexes with p35 or p39 and it is mostly active in the nervous system, regulating several processes. In the past few years, novel roles for Cdk5 have been proposed in many pathological conditions and cancer, even if its role in DLBCL remains uninvestigated to date. For the first time, we showed that Cdk5 and p35 are both overexpressed in DLBCL cell lines. Moreover, we highlighted that proliferation and apoptosis are regulated by Cdk5 activity in loss-of-function experiments. MicroRNAs (miRNAs) are a class of highly conserved short RNAs that regulate diverse cellular processes by degradation or translation inhibition of mRNA targets. Numerous reports have shown that miRNA dysfunction is involved in the development and progression of various human cancers (4). In particular, miR26a has a tumor suppressor role in different cancers, such as nasopharyngeal carcinoma (5) breast cancer (6) and gastric carcinoma (7), but its role in DLBCL remains still unclear. In this study we demonstrated that mir-R26 regulates p35 expression and that it involves Cdk5/p35 pathway directly affecting DLBCL cells proliferation and apoptosis. Thus, the clarification of the molecular mechanisms at the base of Cdk5/p35 expression and their role on DLBCL tumor cell proliferation could lead to the identification of innovative therapeutic targets for treatments of DLBCL

CARATTERIZZAZIONE DELL'ATTIVITA' ANTITUMORALE DELL'INIBITORE DEGLI HDAC ITF2357 (GIVINOSTAT®) IN MODELLI PRECLINICI DI LINFOMA NON-HODGKIN A CELLULE B C-MYC POSITIVI

Il fattore trascrizionale c-Myc gioca un ruolo fondamentale nella patogenesi e nella progressione dei linfomi non-Hodgkin a cellule B (B-NHL) e la sua deregolazione \ue8 generalmente associata ad una cattiva prognosi. L\u2019attivazione aberrante della proteina c-Myc pu\uf2 essere causata da anomalie cromosomiche, come traslocazioni o mutazioni, oppure da modifiche post-traduzionali che ne determinano l\u2019iperespressione. Osservazioni recenti indicano che c-Myc \ue8 in grado non solo di attivare e reprimere rispettivamente molti oncogeni e oncosoppressori, ma anche di modificare il profilo di espressione di alcuni microRNA, stabilendo cos\uec un secondo livello di controllo sull\u2019espressione genica ai fini del mantenimento del fenotipo neoplastico. Date le molteplici evidenze a sostegno della capacit\ue0 di c-Myc di promuovere il suo programma trascrizionale interagendo con regolatori epigenetici, come gli enzimi istone-deacetilasi (HDAC), i farmaci che inibiscono gli HDAC potrebbero essere utilizzati in clinica revertendo il fenotipo neoplastico indotto dall\u2019iperespressione di c-Myc. Pertanto, in questo progetto sono stati analizzati gli effetti antitumorali indotti da un farmaco pan-inibitore degli HDAC, ITF2357 (Givinostat \uae), in modelli preclinici di NHL di tipo B che esprimono ad alti livelli la proteina c-Myc. I risultati ottenuti dimostrano che ITF2357 \ue8 in grado di ridurre significativamente la crescita in vitro di cellule di NHL a cellule B, provocando un arresto del ciclo cellulare in fase G1, accompagnato da un progressivo aumento di apoptosi cellulare. Questi effetti sono correlati alla down-modulazione dei livelli proteici di c-Myc e associati all\u2019induzione di due microRNA che bloccano la sintesi proteica di c-Myc, Let-7a e miR-26a. Dal momento che l\u2019aumento della trascrizione dei microRNA non risulta essere persistente nel tempo e non giustifica pienamente la repressione di c-Myc, sono stati analizzati i livelli di attivazione della traduzione cap-dipendente che regola la sintesi di molte oncoproteine, tra cui anche c-Myc. Il trattamento con ITF2357 inibisce le principali molecole coinvolte nella traduzione cap-dipendente (4E-BP-1, eIF-4E, eIF-4G) e i due maggiori regolatori (PIM e Akt) in funzione della sensibilit\ue0 cellulare all\u2019attivit\ue0 antitumorale dell\u2019HDAC inibitore. Inoltre, il trattamento farmacologico con ITF2357 riduce in modo significativo la crescita in vivo di due modelli cellulari di linfoma di Burkitt, ed in combinazione con ciclofosfamide a dosi sub-ottimali genera in modo sinergico delle remissioni complete dalla malattia nella maggior parte degli animali, al pari o in alcuni casi maggiori rispetto al trattamento con ciclofosfamide a dosi ottimali. Complessivamente, i risultati ottenuti indicano che ITF2357 esercita una spiccata attivit\ue0 antitumorale in linfomi non-Hodgkin a cellule B c-Myc positivi, i quali sono generalmente associati ad una cattiva prognosi. La somministrazione di ITF2357, come singolo agente o in associazione alla chemioterapia convenzionale, potrebbe sia aumentare il tasso di risposte cliniche che ridurre le dosi di chemioterapici limitandone gli effetti collaterali.Recruitment of histone deacetylases (HDACs) by transcription factor c-Myc to promoter regions of regulatory protein- and microRNA-coding genes is essential for its proto-oncogenic functions. Considering the widespread involvement of c-Myc in lymphomagenesis, we studied the antitumor effects of the new-generation pan-HDAC inhibitor ITF2357 (Givinostat\uae) in c- Myc-overexpressing human B-cell non-Hodgkin lymphoma (B-NHL) models. ITF2357 significantly delayed the in vitro growth of B-NHL cell lines by inducing G1 cell-cycle arrest, eventually followed by cell death. These events correlated with the extent of c-Myc protein down-regulation and were associated with the induction of the c-Myc targeting microRNAs let-7a and miR-26a. Since microRNA modulation was not as persistent as c-Myc repression, we investigated whether cap-dependent translation was also affected and found, in effect, that 4E-BP1, eIF4E, and eIF4G, as well as its major positive regulators, Akt and PIM kinases, were inhibited in function of the cell sensitivity to ITF2357 antitumor activity. In vivo, ITF2357 significantly hampered the growth of 2 human Burkitt\u2019s lymphoma models and, in combination with low-dose cyclophosphamide, achieved complete remissions in most animals, equaling or even exceeding the activity of standard cyclophosphamide. Collectively, these findings provide the rationale for testing the clinical advantages of adding ITF2357 to conventional treatments for c-Myc overexpressing lymphomas

SMAC-MIMETIC- AND TRAIL- BASED COMBINATIONS AS THERAPEUTIC STRATEGIES FOR KRAS-MUTATED CANCERS

One third of human cancers are driven by oncogenic mutations in the KRAS loci. In particular, KRAS lesions are predominantly found in the most recalcitrant cancer types of internal organs such as pancreas, colon and lungs. A vast variety of signaling pathways is aberrantly activated downstream mutated KRAS, thus rendering tumours highly aggressive in terms of disease progression, chemoresistance and metastatic potential. Although several attempts have been made to design effective treatment strategies to eradicate these tumours, poor patients\u2019 prognosis still remains a key issue in clinical oncology. One of the main limitations of therapies that selectively target kinase-signaling pathways is the emergence of secondary drug resistance. Therefore, new therapeutic approaches are based on combinatorial therapies, which concomitantly hit the tumour from different sides. Herein, we firstly show a synthetic lethal interaction of Smac-mimetic 83 (SM83) and Camptothecin (CPT) in presence of mutated KRAS employing a model of premalignant tumour cells. Mechanistically, we find that knock-in introduction of mutated KRAS upregulates Noxa via MAPK ERK2. At the same time, SM83, targeting the inhibitors of apoptosis proteins (IAPs) displaces caspase and enhances TNF-mediated cell death. Thus, specific KRAS mutations offer an unusual death-prone scenario in which, together with SM83, CPT efficiently triggers the mitochondrial apoptotic pathway in premalignant cells. Contrarily, in colorectal cancer settings the combination of SM83 and CPT is not lethal when KRAS is mutated suggesting that other pathways are aberrantly activated and render cancer cells resistant to death. Secondly, we propose a novel therapeutic strategy based on selective inhibition of Cyclin Dependent kinase 9 (CDK9) and concomitant induction of apoptosis via death ligand TRAIL in KRAS-mutated non-small cell lung cancer (NSCLC) cell lines. We indeed demonstrate that SNS-032, the most specific and clinically used inhibitor of CDK9, strongly synergizes with TRAIL in killing a panel of human TRAIL-resistant NSCLC cell lines. Moreover, we provide evidence of the efficacy of this novel combination in vivo. In fact, SNS-032 and TRAIL co-treatment totally eradicates established lung tumours. However, as the mice engrafted are immunosuppressed, this model does not provide any information regarding the interaction of the treatment with the murine immune system. Therefore, to further validate the efficacy of our novel combination, we establish an autochthonous mouse model of NSCLC. Preliminary in vitro treatments with TRAIL and CDK9 inhibitor confirm the efficacy of the combination; nevertheless, the setting of the in vivo experiments is still ongoing. Despite several combinatorial approaches have been so far entered clinical trials, patients often relapse and metastasis is still the main cause of cancer-related deaths especially in presence of mutated KRAS, which provides cancer cells with high metastasising potential. Therefore, we finally focus on the mechanisms by which KRAS-mutated tumours metastasize. Indeed, we point out that endogenous TRAIL and TRAIL-R2-mediated signaling is required to promote migration, invasion and metastasis via activation of Rac-1/PI3K signaling axis in KRAS-mutated cancers. Interestingly, we provide genetic in vivo evidence that mouse TRAIL-R is a key driver of cancer progression and metastasis. Significance: KRAS-mutated cancers are still refractory to current targeted therapies. Therefore, we propose two drug-combination approaches, which aim to induce cell death in tumour cells: whereas SM83 in combination with CPT acts at premalignant stages of cancer development, CDK9 inhibition is capable to overcome TRAIL resistance in NSCLC even in vivo, thereby supporting further preclinical investigations. Moreover, we provide new insights on the mechanisms regulating the metastatic process mediated by endogenous TRAIL/TRAIL-R2 pathway. We thus envisage both TRAIL and TRAIL-R2 as potential candidate-targets in clinic

CO-TARGETING OF ONCOGENIC AND DEATH RECEPTORS PATHWAYS IN HUMAN MELANOMA:PRE-CLINICAL RATIONALE FOR A PRO-APOPTOTIC AND ANTI-ANGIOGENIC STRATEGY

INTRODUCTION: Resistance to cell death is one of the well-known hallmarks of cancer. Metastatic melanoma is an aggressive disease whose treatment has significantly improved thanks to the recent development of either target-specific or immune-modulating agents. Since the FDA approval of the BRAFV600E inhibitor Vemurafenib in 2011, several other inhibitors, targeting relevant oncogenic signaling pathways (i.e. the MEK/ERK or the PI3K/Akt/mTOR pathways) have been used to treat melanoma patients. However intrinsic or acquired resistance limits the efficacy of these compounds, with relapses in most of the patients within 6 months. Therefore, there is an immediate necessity for new therapeutic strategies to face this issue, and one option could be represented by combinatorial treatments associating different anti-tumor agents able to target not only tumor cells but also to counteract pro-tumoral mechanisms in melanoma microenvironment. HYPOTHESIS: Our goal was to obtain pre-clinical evidence for the efficacy of an anti-tumor approach based on the combination of MEK, PI3K inhibitors and TRAIL; building upon the hypothesis that these agents should: 1) be able to overcome melanoma intrinsic resistance to programmed cell death by the concomitant targeting of both the extrinsic (mainly through TRAIL activity) and the intrinsic (mainly due to the activity of MEK and PI3K pathway inhibitors) apoptosis pathways; 2) be able to promote an anti-angiogenic effect combining the well-known vascular disrupting activity of TRAIL and the effects of inhibition of pro-angiogenic pathways in tumor and in tumor-associated vasculature due to targeting of ERK and AKT cascades. METHODS: A large panel of patient-derived melanoma cell lines was used to test the in vitro efficacy of the association between AZD6244/Selumetinib (a MEK1/2 inhibitor), the dual PI3K/mTOR inhibitor BEZ235, and soluble TRAIL. Chou-Talalay drug interaction analysis was used to determine Combination Indexes and Fraction Affected values for all the possible combinations of anti-tumor agents. Whole-genome gene expression profiling, flow cytometry experiments, western blot analysis, proteomic arrays and ELISAs were used to clarify the mechanism behind the synergy shown for the AZD6244+TRAIL and AZD6244+BEZ235+TRAIL associations. Moreover, xenografts in SCID mice were used to confirm the in vivo efficacy and mechanism of action of the combination between the MEK inhibitor and TRAIL, using tumor growth rates and immunohistochemistry on tumor nodules to evaluate effects of the association. Human Umbilical Vein Endothelial Cells (HUVEC) were chosen to model the endothelial-melanoma cell interaction in order to analyze its effects on response to the combinatorial treatment, both in terms of apoptosis induction and endothelial differentiation/activation status RESULTS: While half of the melanoma cell lines we tested were resistant to the death receptor ligand TRAIL, several were susceptible either to AZD6244 or to BEZ235, evidencing independent susceptibility profiles to these drugs and setting the rationale for their association. The combination of the MEK inhibitor, with or without the PI3K/mTOR inhibitor, and TRAIL achieved synergistic anti-tumor activity in 20/21 melanoma cell lines tested, including tumors resistant to either one of the agents. Mechanistically, an increment in induction of caspase-dependent cell death and of mitochondrial depolarization was evidenced for the association, and a significant modulation of key regulators of extrinsic and intrinsic apoptosis pathways including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members was confirmed. Moreover, silencing experiments defined Apollon downmodulation as a central event for the promotion of the melanoma apoptotic response to our combinatorial treatments. SCID mice bearing melanoma xenografts were treated with the MEK inhibitor and TRAIL, alone or in combination, obtaining a more significant tumor growth inhibition by the combinatorial treatment, with no detectable adverse events on mice body weight and tissue histology. TUNEL staining on tissues sections indicated also in vivo an increased promotion of tumor apoptosis, which was associated with suppression of several pro-angiogenic molecules like HIF1\uf061, VEGF\uf061, IL-8 and TGF1\uf062\uf020as well as a marked reduction in CD31 positive cells. Furthermore, initial results on HUVECs pointed at a possible effect of the interaction between endothelial and melanoma cells, affecting responsiveness of HUVECs to combinatorial treatments, as documented by increased endothelial cell apoptosis, in response to MEK inhibitor and TRAIL treatment, after co-culture with melanoma cells. The modulatory effect of melanoma on endothelial cells was evidenced not only by \u201cactivation\u201d markers (upregulation of ICAM-1/CD54) but also the \u201cdifferentiation\u201d status of endothelial cells, indicated by increased alpha-SMA levels, reduction in the expression of vascular cadherin CD144 and downmodulation of the endothelial marker CD31. CONCLUSIONS: Results of this work suggest that concomitant targeting of melanoma oncogenic signaling pathways and the TRAIL receptor cascade can not only overcome in vitro tumor resistance to different anti-tumor agents, but can also have in vivo effects on tumor microenvironment, promoting pro-apoptotic effects and inhibition of tumor angiogenesis. Moreover, this could be associated to a modulation of endothelium responsiveness to anti tumor agents by direct interaction with melanoma cells