The use of tyrosine kinase inhibitors (TKI) with activity against ALK, ROS1, or TRKA-C can result in significant clinical benefit in patients with diverse tumors harboring , or rearrangements; however, resistance invariably develops. The emergence of on-target kinase domain mutations represents a major mechanism of acquired resistance. Solvent-front substitutions such as ALK, ROS1 or ROS1, TRKA, and TRKC are among the most recalcitrant of these mechanisms. Repotrectinib (TPX-0005) is a rationally designed, low-molecular-weight, macrocyclic TKI that is selective and highly potent against ROS1, TRKA-C, and ALK. Importantly, repotrectinib exhibits activity against a variety of solvent-front substitutions and As clinical proof of concept, in an ongoing first-in-human phase I/II trial, repotrectinib achieved confirmed responses in patients with or fusion-positive cancers who had relapsed on earlier-generation TKIs due to ROS1 or TRKC solvent-front substitution-mediated resistance. Repotrectinib (TPX-0005), a next-generation ROS1, pan-TRK, and ALK TKI, overcomes resistance due to acquired solvent-front mutations involving , and Repotrectinib may represent an effective therapeutic option for patients with -rearranged malignancies who have progressed on earlier-generation TKIs. .

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave doublestranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.

Elevated interstitial fluid pressure can present a substantial barrier to drug delivery in solid tumors. This is particularly true of pancreatic ductal adenocarcinoma, a highly lethal disease characterized by a robust fibroinflammatory response, widespread vascular collapse, and hypoperfusion that together serve as primary mechanisms of treatment resistance. Free-fluid pressures, however, are relatively low in pancreatic ductal adenocarcinoma and cannot account for the vascular collapse. Indeed, we have shown that the overexpression and deposition in the interstitium of high-molecular-weight hyaluronan (HA) is principally responsible for generating pressures that can reach 100 mmHg through the creation of a large gel-fluid phase. By interrogating a variety of tissues, tumor types, and experimental model systems, we show that an HA-dependent fluid phase contributes substantially to pressures in many solid tumors and has been largely unappreciated heretofore. We investigated the relative contributions of both freely mobile fluid and gel fluid to interstitial fluid pressure by performing simultaneous, real-time fluid-pressure measurements with both the classical wick-in-needle method (to estimate free-fluid pressure) and a piezoelectric pressure catheter transducer (which is capable of capturing pressures associated with either phase). We demonstrate further that systemic treatment with pegylated recombinant hyaluronidase (PEGPH20) depletes interstitial HA and eliminates the gel-fluid phase. This significantly reduces interstitial pressures and leaves primarily free fluid behind, relieving the barrier to drug delivery. These findings argue that quantifying the contributions of free- and gel-fluid phases to hydraulically transmitted pressures in a given cancer will be essential to designing the most appropriate and effective strategies to overcome this important and frequently underestimated resistance mechanism.

p21-activated protein kinase ␥-PAK (Pak2, PAK I) is cleaved by CPP32 (caspase 3) during apoptosis and plays a key role in regulation of cell death. In vitro, CPP32 cleaves recombinant ␥-PAK into two peptides; 1-212 contains the majority of the regulatory domain whereas 213-524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosphorylated with [␥-32 P]ATP. Peptide 1-212 migrates at 27,000 daltons (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 daltons following autophosphorylation on serine (p27P); the catalytic subunit migrates at 34,000 daltons (p34) before and after autophosphorylation on threonine. Following caspase cleavage, a significant lag (ϳ5 min) is observed before autophosphorylation and activity are detected. When ␥-PAK is autophosphorylated with ATP(Mg) alone and then cleaved, only p27 contains phosphate, and the enzyme is inactive with exogenous substrate. After autophosphorylation of ␥-PAK in the presence of Cdc42(GTP␥S) or histone 4, both cleavage products contain phosphate and ␥-PAK is catalytically active. Mutation of the conserved Thr-402 to alanine greatly reduces autophosphorylation and protein kinase activity following cleavage. Thus activation of ␥-PAK via cleavage by CPP32 is a two-step mechanism wherein autophosphorylation of the regulatory domain is a priming step, and activation coincides with autophosphorylation of the catalytic domain.

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous ␥-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant ␥-PAK, while native ␥-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of ␥-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant ␥-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant ␥-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, ␥-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated ␥-PAK, six of which are in common with those previously reported in ␣-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the ␥-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with ␥-PAK activation.

We synthesized a family of sterol-modified glycerophospholipids (SML) in which the sn-1 or sn-2 position is covalently attached to cholesterol and the alternative position contains an aliphatic chain. The SML were used to explore how anchoring cholesterol to a phospholipid affects cholesterol behavior in a bilayer. Notably, cholesterol in the SML retains the membrane condensing properties of free cholesterol regardless of the chemistry or position of its attachment to the glycerol moiety of the phospholipid. SMLs by themselves formed liposomes upon hydration and in mixtures between an SML and diacylglycerophospholipids (C14 to C18 chain length) the thermotropic phase transition is eliminated at the SML equivalent of about 30 mole percent free cholesterol. Osmotic-induced contents leakage from SML (C14-C18) liposomes depends upon the linkage and position of cholesterol but in general is similar to that observed in diacylphosphatidylcholine/ cholesterol: 3/2 (mole ratio) liposomes. SML liposomes are exceptionally resistant to contents release in the presence of serum at 37 °C. This is probably due to fact that SML exchange between bilayers is more than 100 fold less than the exchange rate of free cholesterol in the same conditions. Importantly SML liposomes containing doxorubicin are as effective in treating the murine C26 colon carcinoma, as Doxil™ a commercial liposome doxorubicin formulation. SMLs stabilize bilayers but do not exchange hence provide a new tool for biophysical studies on membranes and they may improve liposomal drug delivery in organs predisposed to the extraction of free cholesterol from bilayers, such as; the skin, lung or blood.

Despite tremendous progress in cancer immunotherapy for solid tumors, clinical success of monoclonal antibody (mAb) therapy is often limited by poorly understood mechanisms associated with the tumor microenvironment (TME). Accumulation of hyaluronan (HA), a major component of the TME, occurs in many solid tumor types, and is associated with poor prognosis and treatment resistance in multiple malignancies. In this study, we describe that a physical barrier associated with high levels of HA (HA high ) in the TME restricts antibody and immune cell access to tumors, suggesting a novel mechanism of in vivo resistance to mAb therapy. We determined that approximately 60% of HER2 3þ primary breast tumors and approximately 40% of EGFR þ head and neck squamous cell carcinomas are HA high , and hypothesized that HA high tumors may be refractory to mAb therapy. We found that the pericellular matrix produced by HA high tumor cells inhibited both natural killer (NK) immune cell access to tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Depletion of HA by PEGPH20, a pegylated recombinant human PH20 hyaluronidase, resulted in increased NK cell access to HA high tumor cells, and greatly enhanced trastuzumab-or cetuximab-dependent ADCC in vitro. Furthermore, PEGPH20 treatment enhanced trastuzumab and NK cell access to HA high tumors, resulting in enhanced trastuzumab-and NK cell-mediated tumor growth inhibition in vivo. These results suggest that HA high matrix in vivo may form a barrier inhibiting access of both mAb and NK cells, and that PEGPH20 treatment in combination with anticancer mAbs may be an effective adjunctive therapy for HA high tumors.

Of the PEG-orthoester lipids tested, POD 2000 is the better choice for the preparation of sterically stabilized NLPs with a small particle diameter, good stability, low cytotoxicity, and satisfactory transfection activity.