Autophagy occurs in tumor cells acquiring cytotoxic drug resistance and its activation may impair their susceptibility to apoptosis in response to apoptogen agents. We investigated the pro-apoptotic effect of dexamethasone (Dex) on MM cell lines (U266, INA-6, LR5-8226, LIG, and MCC2) and primary malignant plasma cells from naïve and refractory/relapsed patients. We evaluated the transcriptional and ultrastructural events leading to autophagy by measuring Beclin-1 and p62 levels and transmission electronic microscopy. Autophagy was inhibited by hydroxychloroquine (HCQ), whereas the ability of Dex-resistant MM cells to recover the susceptibility to apoptosis was measured. A direct relationship between autophagy and Beclin-1 or LC3/Atg8 levels was observed, whereas their mRNAs were inversely correlated to p62 expression. Starvation strongly activated autophagy by inducing cellular, transcriptional, and ultrastructural modifications that were reversed by HCQ. Taken together, these data suggest that autophagy is a potential mechanism leading to drug resistance in MM, and suggest Beclin-1 and p62 as early markers of cell susceptibility to apoptosis. The combination of HCQ with novel agents may thus be considered to improve the therapeutic response in relapsed/resistant MM patients.

Melphalan has been a mainstay of multiple myeloma (MM) therapy for many years. However, following treatment with this alkylator, malignant plasma cells usually escape both apoptosis and cell cycle control, and acquire drug-resistance resulting in tumor progression. Bendamustine is being used inMMpatients refractory to conventional DNA-damaging agents, although the mechanisms driving this lack of cross-resistance are still undefined. Here, we investigated the molecular pathway of bendamustine-induced cell death in melphalan-sensitive and melphalan-resistant MM cell lines. Bendamustine affected cell survival resulting in secondary necrosis, and prompted cell death primarily through caspase-2 activation. Also, bendamustine blocked the cell cycle in the G2/M phase and induced micronucleation, erratic chromosome spreading and mitotic spindle perturbations in melphalan-resistant MM cells. In these cells, both Aurora kinase A (AURKA) and Polo-like kinase-1 (PLK-1), key components of the spindle-assembly checkpoint,were down-regulated following incubationwith bendamustine, whereas levels of Cyclin B1 increased as a consequence of the prolonged mitotic arrest induced by the drug. These findings indicate that, at least in vitro, bendamustine drives cell death by promoting mitotic catastrophe in melphalan-resistantMMcells. Hence, activation of this alternative pathway of cell death may be a novel approach to the treatment of apoptosis-resistant myelomas.

Background: Breast cancer (BC) cells secrete soluble factors that accelerate osteoclast (OC) differentiation, leading to the formation of osteolytic bone metastases. In the BOLERO-2 trial, BC patients with bone involvement who received Everolimus had a delayed tumor progression in the skeleton as a result of direct OC suppression through the inhibition of mTOR, in addition to the general suppressor effect on the cancer cells. Here, we explored the effect of Everolimus, as mTOR inhibitor, on the pro-OC paracrine activity of BC cells. Methods: Both MDA-MB-231 and MCF-7 BC cell lines were incubated with sub-lethal amounts of Everolimus, and their conditioned supernatants were assessed for their capacity to differentiate OCs from PBMC from healthy donors, as well as to interfere with their bone resorbing activity shown on calcium phosphate slices. We also measured the mRNA levels of major pro-OC factors in Everolimus-treated BC cells and their secreted levels by ELISA, and evaluated by immunoblotting the phosphorylation of transcription factors enrolled by pathways cooperating with the mTOR inhibition. Finally, the in vivo pro-OC activity of these cells was assessed in SCID mice after intra-tibial injections. Results: We found that Everolimus significantly inhibited the differentiation of OCs and their in vitro bone-resorbing activity, and also found decreases of both mRNA and secreted pro-OC factors such as M-CSF, IL-6, and IL-1β, whose lower ELISA levels paralleled the defective phosphorylation of NFkB pathway effectors. Moreover, when intra-tibially injected in SCID mice, Everolimus-treated BC cells produced smaller bone metastases than the untreated cells. Conclusions: mTOR inhibition in BC cells leads to a suppression of their paracrine pro-OC activity by interfering with the NFkB pathway; this effect may also account for the delayed progression of bone metastatic disease observed in the BOLERO-2 trial. Keywords: BOLERO-2 trial, Breast cancer cells, mTOR, Osteoclastogenesis, Everolimus

Lenalidomide (LEN) is a structural analogue of Thalidomide and is currently considered a promising compound among immunomodulatory drugs. Following the demonstration of its potent anti-angiogenic, anti-inflammatory, and antineoplastic effects in preclinical models, LEN has emerged as an interesting option for the management of selective hematologic malignancies and may also have a possible role in certain solid tumors as well. It is currently approved in the second-line therapy of multiple myeloma (MM) as well as in myelodysplastic syndrome characterized by 5q minus abnormalities. LEN has been found to be effective in the treatment of both of these conditions and to possess a manageable toxicity profile. In MM, a number of ongoing clinical trials are defining its role in the treatment of newly diagnosed disease as well as in maintenance therapy. Combination approaches pretransplant have shown great promise. Its role in the management of relapsed and refractory disease is now well established. Its long-term tolerability profile appears favorable although an increased risk in new malignancies in patients receiving LEN as maintenance post-stem cell transplant warrants some caution, with follow-up studies being important in determining the long-term implications of this observation.

Langerhans cell histiocytosis (LCH) is a rare proliferative disorder characterized by an accumulation of cells sharing the major phenotypic features of cutaneous Langerhans cells. Given its variable clinical evolution, ranging from self-limiting lesions to multisystemic forms with a poor prognosis, in the last decades it has been debated whether LCH might not have a neoplastic rather than an inflammatory nature. However, although the fundamental events underlying the pathogenesis of LCH are still elusive, recent advances have strikingly improved our understanding of the disease. In particular, the identification of multiple interplays between LCH cells and their tumor microenvironment, along with the recognition of the lesional cytokine storm as a key determinant of LCH progression, has substantiated new opportunities for devising targeted therapeutic approaches. Strikingly, the detection of the rapidly accelerated fibrosarcoma isoform BV600E gain-of-function mutation as a genetic alteration recurring in more than 50% of patients has fueled the paradoxical picture of LCH as a tumor of the antigen-presenting cells that can evade rejection by the immune system.Thus, new evidence regarding the ontogeny of LCH cells, as well as a better understanding of the putative immune system frustrating strategy in LCH, may help to define the precise pathogenesis.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are characterized by mutations of KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) or PDGFRA (platelet-derived growth factor receptor α) that may be efficiently targeted by tyrosine kinase inhibitors (TKI). Notwithstanding the early responsiveness to TKI, the majority of GISTs progress, imposing the need for alternative therapeutic strategies. DOG1 (discovered on GIST-1) shows a higher sensitivity as a diagnostic marker than KIT, however its prognostic role has been little investigated. METHODS: We evaluated DOG1 expression by immunohistochemistry (IHC) in 59 patients with GISTs, and correlated its levels with clinical and pathological features as well as mutational status. Kaplan-Meier analysis was also applied to assess correlations of the staining score with patient recurrence-free survival (RFS). RESULTS: DOG1 was expressed in 66 % of CD117(+) GISTs and highly associated with tumor size and the rate of wild-type tumors. Kaplan-Meier survival analysis showed that a strong DOG1 expression demonstrated by IHC correlated with a worse 2-year RFS rate, suggesting its potential ability to predict GISTs with poor prognosis. CONCLUSIONS: These findings suggest a prognostic role for DOG1, as well as its potential for inclusion in the criteria for risk stratification.

Neuroendocrine tumors (NETs) metastasize to the bone. However, the incidence, clinical features, management and pathogenesis of bone involvement in NET patients have been poorly investigated.

The therapeutic landscape of gastroenteropancreatic-neuroendocrine tumors (GEP-NETs) has evolved significantly in recent years. Current and emerging treatment options include somatostatin analogs, radiolabeled somatostatin analogs, the mTOR inhibitor everolimus, and the tyrosine kinase inhibitor sunitinib. Although high-quality data from phase III trials are lacking, cytotoxic agents are commonly used for the treatment of poorly differentiated neuroendocrine carcinomas and well-differentiated NETs originating in the pancreas. Hepatic-directed therapies are recommended for patients with slow-growing, liver-predominant disease but have never been compared to systemic agents. Telotristat ethyl, a novel serotonin synthesis inhibitor, has recently demonstrated efficacy in palliating diarrhea in patients with poorly controlled carcinoid syndrome. In the absence of definite predictive biomarkers, therapeutic decisions in most cases rely on clinical and pathological criteria. However, navigating the current therapeutic algorithm may be challenging, and future trials need to address several important questions: what is the best sequence of treatment? Is there a role for combination therapies in GEP-NETs? Are neoadjuvant, adjuvant, or maintenance strategies safe and effective? Do all NET patients require active treatment? What new molecular targets can be clinically exploited? A tight integration between basic and clinical research is needed to further advance the field of NETs.