Silvia L. Locatelli

Sorafenib Inhibits Lymphoma Xenografts by Targeting MAPK/ERK and AKT Pathways in Tumor and Vascular Cells

The anti-lymphoma activity and mechanism(s) of action of the multikinase inhibitor sorafenib were investigated using a panel of lymphoma cell lines, including SU-DHL-4V, Granta-519, HD-MyZ, and KMS-11 cell lines. In vitro, sorafenib significantly decreased cell proliferation and phosphorylation levels of MAPK and PI3K/Akt pathways while increased apoptotic cell death. In vivo, sorafenib treatment resulted in a cytostatic rather than cytotoxic effect on tumor cell growth associated with a limited inhibition of tumor volumes. However, sorafenib induced an average 50% reduction of tumor vessel density and a 2-fold increase of necrotic areas. Upon sorafenib treatment, endothelial and tumor cells from SU-DHL-4V, Granta-519, and KMS-11 nodules showed a potent inhibition of either phospho-ERK or phospho-AKT, whereas a concomitant inhibition of phospho-ERK and phospho-AKT was only observed in HD-MyZ nodules. In conclusion, sorafenib affects the growth of lymphoid cell lines by triggering antiangiogenic mechanism(s) and directly targeting tumor cells.

Phase II study of sorafenib in patients with relapsed or refractory lymphoma.

The safety and activity of the multikinase inhibitor sorafenib were investigated in patients with relapsed or refractory lymphoproliferative disorders who received sorafenib (400 mg) twice daily until disease progression or appearance of significant clinical toxicity. The primary endpoint was overall response rate (ORR). Biomarkers of sorafenib activity were analysed at baseline and during treatment. Thirty patients (median age, 61 years; range, 18–74) received a median of 4 months of therapy. Grade 3–4 toxicities included hand/foot skin reactions (20%), infections (12%), neutropenia (20%) and thrombocytopenia (14%). Two patients achieved complete remission (CR), and two achieved partial remission (PR) for an ORR of 13%. Stable disease (SD) and progressive disease (PD) was observed in 15 (50%) and 11 patients (37%), respectively. The median overall survival (OS) for all patients was 16 months. For patients who achieved CR, PR and SD, the median time to progression and OS was 5 and 24 months, respectively. Compared with patients with PD, responsive patients had significantly higher baseline levels of extracellular signal‐regulated kinase phosphorylation and autophagy and presented a significant reduction of these parameters after 1 month of therapy. Sorafenib was well tolerated and had a clinical activity that warrants development of combination regimens.

Perifosine and sorafenib combination induces mitochondrial cell death and antitumor effects in NOD/SCID mice with Hodgkin lymphoma cell line xenografts

The effects of the Akt inhibitor perifosine and the RAF/MEK/ERK inhibitor sorafenib were investigated using two CD30+Hodgkin lymphoma cell lines (L-540 and HDLM-2) and the CD30−HD-MyZ histiocytic cell line. The combined perifosine/sorafenib treatment significantly inhibited mitogen-activated protein kinase and Akt phosphorylation in two of the three cell lines. Profiling of the responsive cell lines revealed that perifosine/sorafenib decreased the amplitude of transcriptional signatures that are associated with the cell cycle, DNA replication and cell death. Tribbles homolog 3 (TRIB3) was identified as the main mediator of the in vitro and in vivo antitumor activity of perifosine/sorafenib. Combined treatment compared with single agents significantly suppressed cell growth (40–80%, P<0.001), induced severe mitochondrial dysfunction and necroptotic cell death (up to 70%, P<0.0001) in a synergistic manner. Furthermore, in vivo xenograft studies demonstrated a significant reduction in tumor burden (P<0.0001), an increased survival time (81 vs 45 days, P<0.0001), an increased apoptosis (2- to 2.5-fold, P<0.0001) and necrosis (2- to 8-fold, P<0.0001) in perifosine/sorafenib-treated animals compared with mice receiving single agents. These data provide a rationale for clinical trials using perifosine/sorafenib combination.

Myeloablative doses of yttrium-90-ibritumomab tiuxetan and the risk of secondary myelodysplasia/acute myelogenous leukemia

Because the long‐term toxicity of myeloablative radioimmunotherapy remains a matter of concern, the authors evaluated the hematopoietic damage and incidence of secondary myelodysplastic syndrome and acute myelogenous leukemia (sMDS/AML) in patients who received myeloablative doses of the radiolabeled antibody yttrium‐90 (90Y)‐ibritumomab tiuxetan.

Phase II Study of Perifosine and Sorafenib Dual-Targeted Therapy in Patients with Relapsed or Refractory Lymphoproliferative Diseases

Purpose: To evaluate safety and activity of perifosine and sorafenib combination therapy in patients with lymphoproliferative diseases. Experimental Design: Patients with relapsed and refractory lymphoproliferative diseases received perifosine (50 mg twice daily) for 1 month. Patients achieving less than partial response (PR) after perifosine alone were administered the combination therapy [perifosine plus sorafenib (400 mg twice daily)] until progressive disease (PD) or unacceptable toxicity occurred. The pERK and pAKT in peripheral blood lymphocytes as well as serum cytokine levels were investigated as predictive biomarkers of response. Results: Forty patients enrolled in this study. After 1 month of perifosine alone, 36 who achieved less than PR went on to combination therapy, whereas four patients with chronic lymphocytic leukemia (CLL) who achieved PR continued with perifosine alone for a median of 10 months (range, 4–21). The most common drug-related toxicities were grade 1–2 anemia (17%), thrombocytopenia (9%), diarrhea (25%), joint pain (22%), and hand–foot skin reaction (25%). Three patients experienced grade 3 pneumonitis. Eight patients (22%) achieved PR, 15 (42%) achieved stable disease, and 13 (36%) experienced PD. A 28% PR rate was recorded for 25 patients with Hodgkin lymphoma. Among all patients, median overall survival and progression-free survival were 16 and 5 months, respectively. Early reductions in pERK and pAKT significantly correlated with the probability of clinical response. Conclusions: Perifosine and sorafenib combination therapy is feasible with manageable toxicity and demonstrates promising activity in patients with Hodgkin lymphoma. The predictive value of pERK and pAKT should be confirmed in a larger patient cohort. Clin Cancer Res; 20(22); 5641–51. ©2014 AACR.

BIM upregulation and ROS-dependent necroptosis mediate the antitumor effects of the HDACi Givinostat and Sorafenib in Hodgkin lymphoma cell line xenografts

Relapsed/refractory Hodgkin’s lymphoma (HL) is an unmet medical need requiring new therapeutic options. Interactions between the histone deacetylase inhibitor Givinostat and the RAF/MEK/ERK inhibitor Sorafenib were examined in HDLM-2 and L-540 HL cell lines. Exposure to Givinostat/Sorafenib induced a synergistic inhibition of cell growth (range, 70–80%) and a marked increase in cell death (up to 96%) due to increased H3 and H4 acetylation and strong mitochondrial injury. Gene expression profiling indicated that the synergistic effects of Givinostat/Sorafenib treatment are associated with the modulation of cell cycle and cell death pathways. Exposure to Givinostat/Sorafenib resulted in sustained production of reactive oxygen species (ROS) and activation of necroptotic cell death. The necroptosis inhibitor Necrostatin-1 prevented Givinostat/Sorafenib-induced ROS production, mitochondrial injury, activation of BH3-only protein BIM and cell death. Knockdown experiments identified BIM as a key signaling molecule that mediates Givinostat/Sorafenib-induced oxidative death of HL cells. Furthermore, in vivo xenograft studies demonstrated a 50% reduction in tumor burden (P<0.0001), a 5- to 15-fold increase in BIM expression (P⩽0.0001) and a fourfold increase in tumor necrosis in Givinostat/Sorafenib-treated animals compared with mice that received single agents. These results provide a rationale for exploring Givinostat/Sorafenib combination in relapsed/refractory HL.

Constitutive localization of DR4 in lipid rafts is mandatory for TRAIL-induced apoptosis in B-cell hematologic malignancies

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts as an apoptosis inducer for cancer cells sparing non-tumor cell targets. However, several phase I/II clinical trials have shown limited benefits of this molecule. In the present work, we investigated whether cell susceptibility to TRAIL ligation could be due to the presence of TRAIL death receptors (DRs) 4 and 5 in membrane microdomains called lipid rafts. We performed a series of analyses, either by biochemical methods or fluorescence resonance energy transfer (FRET) technique, on normal cells (i.e. lymphocytes, fibroblasts, endothelial cells), on a panel of human cancer B-cell lines as well as on CD19+ lymphocytes from patients with B-chronic lymphocytic leukemia, treated with different TRAIL ligands, that is, recombinant soluble TRAIL, specific agonistic antibodies to DR4 and DR5, or CD34+ TRAIL-armed cells. Irrespective to the expression levels of DRs, a molecular interaction between ganglioside GM3, abundant in lymphoid cells, and DR4 was detected. This association was negligible in all non-transformed cells and was strictly related to TRAIL susceptibility of cancer cells. Interestingly, lipid raft disruptor methyl-beta-cyclodextrin abrogated this susceptibility, whereas the chemotherapic drug perifosine, which induced the recruitment of TRAIL into lipid microdomains, improved TRAIL-induced apoptosis. Accordingly, in ex vivo samples from patients with B-chronic lymphocytic leukemia, the constitutive embedding of DR4 in lipid microdomains was associated per se with cell death susceptibility, whereas its exclusion was associated with TRAIL resistance. These results provide a key mechanism for TRAIL sensitivity in B-cell malignances: the association, within lipid microdomains, of DR4 but not DR5, with a specific ganglioside, that is the monosialoganglioside GM3. On these bases we suggest that lipid microdomains could exert a catalytic role for DR4-mediated cell death and that an ex vivo quantitative FRET analysis could be predictive of cancer cell sensitivity to TRAIL.

Autophagy as a pathogenic mechanism and drug target in lymphoproliferative disorders

Autophagy represents a key mechanism of cytoprotection that can be activated by a variety of extracellular and intracellular stresses and allows the cell to sequester cytoplasmic components and damaged organelles, delivering them to lysosomes for degradation and recycling. However, the autophagy process has also been associated with the death of the cell. It has been demonstrated to be constitutive in some instances and inducible in others, and the idea that it could represent a pathogenetic determinant as well as a possible prognostic tool and a therapeutic target in a plethora of human diseases has recently been considered. Among these, cancer represents a major one. In this review, we recapitulate the critical implications of autophagy in the pathogenesis, progression, and treatment of lymphoproliferative disorders. Leukemias and lymphomas, in fact, represent paradigmatic human diseases in which advances have recently been made in this respect.—Pierdominici, M., Barbati, C., Vomero, M., Locatelli, S. L., Carlo‐Stella, C., Ortona, E., Malorni, W. Autophagy as a pathogenic mechanism and drug target in lymphoproliferative disorders. FASEB J. 28, 524–535 (2014). www.fasebj.org

Circulating tumor DNA reveals genetics, clonal evolution and residual disease in classical Hodgkin lymphoma

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL.

Estrogen receptor β ligation inhibits Hodgkin lymphoma growth by inducing autophagy

Although Hodgkin lymphoma (HL) is curable with current therapy, at least 20% of patients relapse or fail to make complete remission. In addition, patients who achieve long-term disease-free survival frequently undergo infertility, secondary malignancies, and cardiac failure, which are related to chemotherapeutic agents and radiation therapies. Hence, new therapeutic strategies able to counteract the HL disease in this important patient population are still a matter of study. Estrogens, in particular 17β-estradiol (E2), have been suggested to play a role in lymphoma cell homeostasis by estrogen receptors (ER) β activation. On these bases, we investigated whether the ligation of ERβ by a selective agonist, the 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), could impact HL tumor growth. We found that DPN-mediated ERβ activation led to a reduction of in vitro cell proliferation and cell cycle progression by inducing autophagy. In nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice engrafted with HL cells, ERβ activation by DPN was able to reduce lymphoma growth up to 60% and this associated with the induction of tumor cell autophagy. Molecular characterization of ERβ-induced autophagy revealed an overexpression of damage-regulated autophagy modulator 2 (DRAM2) molecule, whose role in autophagy modulation is still debated. After ERβ activation, both DRAM2 and protein 1 light chain 3 (LC3), a key actor in the autophagosome formation, strictly interacted each other and localized at mitochondrial level. Altogether these results suggest that targeting ERβ with selective agonists might affect HL cell proliferation and tumor growth via a mechanism that brings into play DRAM2-dependent autophagic cascade.