Panobinostat (pano) is an FDA-approved histone deacetylase inhibitor. There is interest in evaluating alternate dosing schedules and novel combinations of pano for the treatment of upper aerodigestive and lung malignancies; thus we evaluated it in combination with Taxol, a chemotherapeutic with activity in both diseases. Dose-dependent synergy was observed in Non-Small Cell Lung Cancer (NSCLC) and Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines and was due to senescence rather than potentiation of cell death. Senescence occurred following cisplatin- or Taxol-treatment in cell lines from both cancer types and was associated with decreased histone 3 (H3) acetylation and increased Bcl-xL expression: the latter a biomarker of senescence and target of anti-senescence therapeutics, or senolytics. Since H3 acetylation and Bcl-xL expression were altered in senescence, we subsequently evaluated pano as a senolytic in chemotherapy-treated cancer cells enriched for senescent cells. Pano caused cell death at significantly higher rates compared to repeat dosing with chemotherapy. This was associated with decreased expression of Bcl-xL and increased acetylated H3, reversing the expression patterns observed in senescence. These data support evaluating pano as a post-chemotherapy senolytic with the potential to kill persistent senescent cells that accumulate during standard chemotherapy in NSCLC and HNSCC.

Intracellular membrane trafficking plays an essential role in the biogenesis and maintenance of myelin. Members of the Rab protein family are important components of the systems that regulate intracellular vesicle transport. We examine the function of rRab22b, a novel rat Rab protein cloned from an oligodendrocyte cDNA library, by visualizing and identifying in living Hela cells the organelles that contain rRab22b. Our results show that rRab22b is present in the trans Golgi/TGN and endocytic compartments. Trafficking of membranes from trans Golgi to endocytic compartments takes place via small tubulo vesicular organelles containing rRab22b. The formation of vesicles in the trans Golgi also appears to be regulated by rRab22b. Additionally, our results suggest that rRab22b controls the transport of vesicles from the trans Golgi to endocytic compartments that localize in oligodendrocyte processes. That rRab22b is involved in the transport of certain proteins from trans Golgi to myelin is suggested by the evidence that certain proteins being targeted to the plasma membrane are first transported from trans Golgi to endocytic compartments.

Rab31, a protein that we originally cloned from a rat oligodendrocyte cDNA library, localizes in the trans Golgi network (TGN) and endosomes. However, its function has not yet been established. Here we show the involvement of Rab31 in the transport of mannose 6-phosphate receptors (MPRs) from TGN to endosomes. We demonstrate the specific sorting of cation-dependent-MPR (CD-MPR), but not CD63 and vesicular stomatitis virus G (VSVG) protein, to Rab31-containing trans-Golgi network carriers. CD-MPR and Rab31 containing carriers originate from extending TGN tubules that also contain clathrin and GGA1 coats. Expression of constitutively active Rab31 reduced the content of CD-MPR in the TGN relative to that of endosomes, while expression of dominant negative Rab31 triggered reciprocal changes in CD-MPR distribution. Expression of dominant negative Rab31 also inhibited the formation of carriers containing CD-MPR in the TGN, without affecting the exit of VSVG from this compartment. Importantly, siRNA-mediated depletion of endogenous Rab31 caused collapse of the Golgi apparatus. Our observations demonstrate that Rab31 is required for transport of MPRs from TGN to endosomes and for the Golgi/TGN organization.

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Intracellular membrane trafficking plays an essential role in the structural and functional organization of oligodendrocytes, which synthesize a large amount of membrane to form myelin. Rab proteins are key components in intracellular vesicular transport. We cloned a novel Rab protein from an oligodendrocyte cDNA library, designating it Rab40c because of its homology with Rab40a and Rab40b. The DNA sequence of Rab40c shows an 843-base pair open reading frame. The deduced amino acid sequence is a protein with 281 amino acids, with a molecular weight of 31,466 Da and an isoelectric point of 9.83. Rab40c presents a number of distinct structural features including a carboxyl terminal extension and amino acid substitutions in the consensus sequence of the GTP-binding motifs. The carboxyl terminal region contains motifs that permit isoprenylation and palmitoylation. Binding studies indicate that Rab40c binds guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S) with a K(d) of 21 microM and has a higher affinity for guanosine triphosphate (GTP) than for guanosine diphosphate (GDP). Rab40c is localized in the perinuclear recycling compartment, suggesting its involvement in endocytic events such as receptor recycling. The importance of this recycling in myelin formation is suggested by the increase in both Rab40c mRNA and Rab40c protein as oligodendrocytes differentiate.

While the clinical benefit of MEK inhibitor (MEKi)-based therapy is well established in Raf mutant malignancies, its utility as a suppressor of hyperactive MAPK signaling in the absence of mutated Raf or Ras, is an area of ongoing research. MAPK activation is associated with loss of ERα expression and hormonal resistance in numerous malignancies. Herein, we demonstrate that MEKi induces a feedback response that results in ERα overexpression, phosphorylation and transcriptional activation of ER-regulated genes. Mechanistically, MEKi-mediated ERα overexpression is largely independent of erbB2 and AKT feedback activation, but is ERK-dependent. We subsequently exploit this phenomenon therapeutically by combining the ER-antagonist, fulvestrant with MEKi. This results in synergistic suppression of tumor growth, in vitro and potentiation of single agent activity in vivo in nude mice bearing xenografts. Thus, we demonstrate that exploiting adaptive feedback after MEKi can be used to sensitize ERα-positive tumors to hormonal therapy, and propose that this strategy may have broader clinical utility in ERα-positive ovarian carcinoma.

Rab31, a protein that we cloned from an oligodendrocyte cDNA library, is required for transport of mannose 6-phosphate receptors (MPRs) from the TGN to endosomes and for the Golgi/TGN organization. Here we extend the knowledge of the mechanism of action of Rab31 by demonstrating its interaction with OCRL-1, a phosphatidylinositol 4, 5 diphosphate 5-phosphatase (PI(4,5)P2 5-phosphatase) that regulates the levels of PI(4,5)P2 and PI(4)P, molecules involved in transport and Golgi/TGN organization. We show that Rab31 interacts with OCRL-1 by yeast two hybrid system, GST-Rab31 pull-down experiments and coimmunoprecipitation of OCRL-1 using oligodendrocyte culture lysates. Rab31 and OCRL-1 colocalize in the TGN, post TGN carriers and endosomes. Cation-dependent-MPR (CD-MPR) is sorted to OCRL-1-containing carriers, but CD63 and vesicular stomatitis virus G (VSVG) are not. siRNA-mediated depletion of endogenous Rab31 causes collapse of the TGN apparatus and markedly decreases the levels of OCRL-1 in the TGN and endosomes. Our observations indicate that the role of Rab31 in the Golgi/TGN structure and transport of MPRs depends on its capability to recruit OCRL-1 to domains of the TGN where the formation of carriers occurs. The importance of our observations is highlighted by the fact that mutation of OCRL-1 causes demyelination in humans.

Intracellular membrane trafficking plays an essential role in the biogenesis and maintenance of myelin. Members of the Rab protein family are important components of the systems that regulate intracellular vesicle transport. We examine the function of rRab22b, a novel rat Rab protein cloned from an oligodendrocyte cDNA library, by visualizing and identifying in living Hela cells the organelles that contain rRab22b. Our results show that rRab22b is present in the trans Golgi/TGN and endocytic compartments. Trafficking of membranes from trans Golgi to endocytic compartments takes place via small tubulo vesicular organelles containing rRab22b. The formation of vesicles in the trans Golgi also appears to be regulated by rRab22b. Additionally, our results suggest that rRab22b controls the transport of vesi-cles from the trans Golgi to endocytic compartments that localize in oligodendrocyte processes. That rRab22b is involved in the transport of certain proteins from trans Golgi to myelin is suggested by the evidence that certain proteins being targeted to the plasma membrane are first transported from trans Golgi to endocytic compartments.