Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, is an important regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER is reorganized. Knocking out both FAM134B isoforms in myotubes results in an aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine-tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells.

Background: Given the consistent antitumor activity and favorable toxicity profile in heavily pretreated patients, BDM represents a suitable platform for combination with target-based agents in the setting of recurrent HL. The anti-CD30 antibody-monomethyl auristatin E (MMAE) conjugate brentuximab vedotin (BV) appears a most valuable candidate by coupling an impressive clinical activity with the lack of serious overlapping toxicities with BDM. While the combination of BDM and BV is being evaluated in Phase II studies, no information is available as to possible mechanisms through which BDM may regulate the antitumor efficacy of BV towards HL cells. Methods: We evaluated the effects of acute and extended exposure to BDM on CD30 expression and sensitivity to the cytotoxic activity BV in the HL cell line L1236. Cells were cultured in the presence of BDM at its IC50 for different time points of acute exposure. Through continuous exposure to increasing concentrations (25.0, 50.0, 75.0, 100 micromol/L) of BDM, we then performed a serial in vitro selection for BDM-resistant (R) L1236 cell clones and determined their CD30 expression by flow cytometry, qRT-PCR, and Western analysis. Results: Acute exposure to BDM (48 to 72 hrs) of L1236 cells led to a sizeable upregulation of CD30 as shown by flow cytometry. This effect was unsustained since CD30 intensity returned to baseline levels upon culturing in BDM-free medium for one week. Expression of other surface antigens, i.e. HLA-DR, was unaffected by BDM while acute exposure to doxorubicin and other cytotoxic drugs did not modify the CD30 expression. We established four L1236-derived cell clones (R25, R50, R75 and R100) able to proliferate across the different concentrations of BDM with growth/viability curves superimposable to native cells. Clonal identity among clones and with parental cells was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions. All R-clones displayed an up to 900% increase in CD30 median fluorescence intensity, relative to native L1236 cells (Figure 1A). The sustained CD30 upregulation was confirmed by qRT-PCR and Western blotting since R-clones expressed up to 10-fold higher levels of CD30-specific mRNA and CD30-specific 120 kDa components as compared to parental cells. To rule out a general upregulation of surface molecules as a result of the chronic exposure to BDM we evaluated the expression of other surface antigens. As opposed to CD30, HLA-DR and PDL-1 relative transcript levels and cell surface expression were significantly reduced in R-cells. Intriguingly, BDM-induced CD30 upregulation was specific to L1236 cells since extended exposure to BDM did not modify expression levels of CD30 in other lymphoma cell types (SUDHL1, JEKO). BDM-induced CD30 overexpression significantly enhanced the sensitivity of HL cells to the cytotoxic effects of BV. MTS assay showed the IC50 of R100 cells to BV shifted to 0.21 ± 0.06 mcg/mL (48 hrs) and 0.19 ± 0.05 mcg/mL (72 hrs) vs. 3.16 ± 0.75 mcg/mL (48 hrs) and 3.87 ± 0.68 mcg/mL (72 hrs) of parental cells, a 15- and 20-fold increase, respectively (p<0.001; Figure 1B). Finally, qRT-PCR studies disclosed that R-clones did not show any upregulation of the drug exporter MDR1 gene, involved in MMAE extrusion and BV resistance in HL cells, as compared to parental L1236 cells. Conclusions: Whileacute exposure to BDM induced a transient upregulation of CD30, extended exposure to the agent resulted in the selection of L1236 subclones with a stable overexpression of surface CD30 and reduced levels of HLA-DR and PDL-1. Upregulation of CD30 was associated with a significantly enhanced sensitivity to cytotoxic effects of BV. Translated into a clinical context these findings suggest that HL patients progressing upon BDM treatment may turn exquisitely sensitive to BV and prompt the exploration of a sequential treatment with BDM and BV in late and earlier disease phases. Results also provide a mechanistic insight as to the efficacy of the BDM-BV combination in HL, highlighting non-overlapping resistance pathways between BDM and MMAE. BDM-induced selection of CD30^hi, HLA-DR^low/PDL-1^low tumor cell clones might be relevant to the design of novel treatment strategies for HL based on the combination of BDM with BV and anti-PD1/PDL-1 antibodies.

NGAL promotes recruitment of tumor infiltrating leukocytes NGAL promotes recruitment of tumor infiltrating leukocytes

The transforming growth factor beta (TGF-β) signaling is fundamental for correct embryonic development. However, alterations of this pathway have been correlated with oncogenesis, tumor progression and sustaining of cancer stem cells (CSCs). Cripto-1 (CR-1) and Nodal are two embryonic proteins involved in TGF-β signaling. Their expression is almost undetectable in terminally differentiated cells, but they are often re-expressed in tumor cells, especially in CSCs. Moreover, cancer cells that show high levels of CR-1 and/or Nodal display more aggressive phenotypes in vitro, while in vivo their expression correlates with a worse prognosis in several human cancers. The ability to target CSCs still represents an unmet medical need for the complete eradication of certain types of tumors. Given the prognostic role and the selective expression of CR-1 and Nodal on cancer cells, they represent archetypes for targeted therapy. The aim of this review is to clarify the role of CR-1 and Nodal in cancer stem populations and to summarize the current therapeutic strategy to target CSCs using monoclonal antibodies (mAbs) or other molecular tools to interfere with these two proteins.

Background: The highly unfavorable outcome of patients with recurrent HL, who progress after stem cell transplantation or are ineligible for such procedure make the development of new active agents an impellent medical need in this clinical setting. EDO-S101 is fusion molecule combining the DNA damaging effects of bendamustine (BDM) with the pan-histone deacetylase (HDAC) inhibitor, vorinostat. Given that BDM and HDAC inhibitors are active agents in recurrent HL we investigated the preclinical activity of EDO-S101 in this malignancy. Methods: We assessed the patterns of EDO-S101 cytotoxicity (0.39 to 50 µmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its regulatory effects on genes involved in DNA-damage/repair response, apoptosis and cell cycle checkpoints. As a further model we exploited an L1236 cell clone (R100) selected for resistance (R) to BDM through continuous exposure to increasing concentrations of the agent. R100 cells display a growth pattern indistinguishable from parental L1236 cells when cultured in the presence of BDM (100 µmol/L). Clonal identity of R100 cells with parental L1236 was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions. Results: EDO-S101 induced a significant time- and dose-dependent inhibition of growth and survival in all HL cell lines. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 1.88 µmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 2.06, 2.53, 2.26 and 16.2 µmol/L, respectively. These values were about 10-fold lower than the IC50 of BDM in the same cell lines. While exposure of L1236 cells to EDO-S101 caused cell accumulation in S-phase, qRT-PCR disclosed that cell death was mainly dependent on triggering of apoptosis, as shown by the early (24 hrs) and sustained (48 hrs) upregulation of NOXA, p21 and p27 genes. Data were confirmed by the significant increase (>150%) of Annexin V-expressing L1236 cells. In contrast, expression levels of PLK1, AKA and cyclin B1 genes remained unchanged or were increased. This excluded induction of the mitotic catastrophe (MC) as a major determinant of cytotoxic activity for EDO-S101 in L1236 cells. Exposure to EDO-S101 induced a strong DNA stress/repair response as shown by the activation of pATR/pATM and increase of the downstream DNA damage checkpoint proteins pCHK1-/-2 and CCNB1, along with the upregulation of the EXO1 gene. Most intriguingly, BDM-resistant L1236 cells (R100) were highly sensitive to EDO-S101, with an IC50 of only 4.56 µmol/L, but less responsive to vorinostat (IC50: 6.17 µmol/L) than parental L1236 cells (IC50: 0.58 µmol/L). Differently from native cells, EDO-S101 induced a late downregulation of transcripts for PLK1 and AKA genes and of cyclin B1 gene and protein in R100 cells, along with the early induction of NOXA and p21, but not p27 genes. In both L1236 and R100 cells, expression of MC-genes was unaffected by exposure to vorinostat. This suggests a more complex mechanism for EDO-S101 in BDM-resistant HL cells involving activation of the both apoptotic and MC pathways. Notably, we documented that EDO-S101 corrected the constitutive ATM/ATR unbalance of R100 cells by triggering the early (24 hrs) upregulation of ATR and a late (48 hrs) downregulation of ATM transcripts and proteins, along with increased levels of EXO1 and MGMT at 24 hrs. Vorinostat induced a similar effect. Finally, while baseline expression levels of HDAC isoforms were comparable among HL cell lines, EDO-S101 caused a significant (>40%) late downregulation of transcripts for all HDAC isoforms (HDAC-1 to -8) in R100 cells but only of HDAC-6 in native L1236. This pattern diverged from results obtained in both L1236, i.e. increase of all HDAC isoform transcripts except HDAC-6, and R100 cells, i.e. upregulation of all isoforms and reduction of HDAC-6, with vorinostat and BDM as single agents. Conclusions: We have described for the first time that EDO-S101 is effective in preclinical models of HL including cells resistant to BDM. The combined functions of in one molecule of a bifunctional alkylator and panHDAC inhibitor confer this agent unique antitumor property different from both of its single drug components. Following a strong DNA damage response, triggering of apoptosis and/or MC may take place in HL cells according to their sensitivity status to BDM. A phase 1/2 study in recurrent HL, including patients pretreated with BDM, is next to be launched.