Histone demethylase KDM5D upregulation drives sex differences in colon cancer.

Sex exerts a profound impact on cancer incidence, spectrum and outcomes, yet the molecular and genetic bases of such sex differences are ill-defined and presumptively ascribed to X-chromosome genes and sex hormones1. Such sex differences are particularly prominent in colorectal cancer (CRC) in which men experience higher metastases and mortality. A murine CRC model, engineered with an inducible transgene encoding oncogenic mutant KRASG12D and conditional null alleles of Apc and Trp53 tumour suppressors (designated iKAP)2, revealed higher metastases and worse outcomes specifically in males with oncogenic mutant KRAS (KRAS*) CRC. Integrated cross-species molecular and transcriptomic analyses identified Y-chromosome gene histone demethylase KDM5D as a transcriptionally upregulated gene driven by KRAS*-mediated activation of the STAT4 transcription factor. KDM5D-dependent chromatin mark and transcriptome changes showed repression of regulators of the epithelial cell tight junction and major histocompatibility complex class I complex components. Deletion of Kdm5d in iKAP cancer cells increased tight junction integrity, decreased cell invasiveness and enhanced cancer cell killing by CD8+ T cells. Conversely, iAP mice engineered with a Kdm5d transgene to provide constitutive Kdm5d expression specifically in iAP cancer cells showed an increased propensity for more invasive tumours in vivo. Thus, KRAS*-STAT4-mediated upregulation of Y chromosome KDM5D contributes substantially to the sex differences in KRAS* CRC by means of its disruption of cancer cell adhesion properties and tumour immunity, providing an actionable therapeutic strategy for metastasis risk reduction for men afflicted with KRAS* CRC.

Lentiviral interferon: A novel method for gene therapy in bladder cancer.

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia.

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.

A randomized, double-blind, placebo-controlled study of daily cannabidiol for the treatment of canine osteoarthritis pain.

ABSTRACT: Over the last 2 decades, affirmative diagnoses of osteoarthritis (OA) in the United States have tripled due to increasing rates of obesity and an aging population. Hemp-derived cannabidiol (CBD) is the major nontetrahydrocannabinol component of cannabis and has been promoted as a potential treatment for a wide variety of disparate inflammatory conditions. Here, we evaluated CBD for its ability to modulate the production of proinflammatory cytokines in vitro and in murine models of induced inflammation and further validated the ability of a liposomal formulation to increase bioavailability in mice and in humans. Subsequently, the therapeutic potential of both naked and liposomally encapsulated CBD was explored in a 4-week, randomized placebo-controlled, double-blinded study in a spontaneous canine model of OA. In vitro and in mouse models, CBD significantly attenuated the production of proinflammatory cytokines IL-6 and TNF-α while elevating levels of anti-inflammatory IL-10. In the veterinary study, CBD significantly decreased pain and increased mobility in a dose-dependent fashion among animals with an affirmative diagnosis of OA. Liposomal CBD (20 mg/day) was as effective as the highest dose of nonliposomal CBD (50 mg/day) in improving clinical outcomes. Hematocrit, comprehensive metabolic profile, and clinical chemistry indicated no significant detrimental impact of CBD administration over the 4-week analysis period. This study supports the safety and therapeutic potential of hemp-derived CBD for relieving arthritic pain and suggests follow-up investigations in humans are warranted.

Glioblastoma stem cell-derived exosomes induce M2 macrophages and PD-L1 expression on human monocytes.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Imaging Intratumoral Nanoparticle Uptake After Combining Nanoembolization with Various Ablative Therapies in Hepatic VX2 Rabbit Tumors.

Combining image-guided therapy techniques for the treatment of liver cancers is a strategy that is being used to improve local tumor control rates. Here, we evaluate the intratumoral uptake of nanoparticles used in combination with radiofrequency ablation (RFA), irreversible electroporation (IRE), or laser induced thermal therapy (LITT). Eight rabbits with VX2 tumor in the liver underwent one of four treatments: (i) nanoembolization (NE) with radiolabeled, hollow gold nanoparticles loaded with doxorubicin (64Cu-PEG-HAuNS-DOX); (ii) NE + RFA; (iii) NE + IRE; (iv) NE +LITT. Positron emission tomography/computed tomography (PET/CT) imaging was obtained 1-hr or 18-hrs after intervention. Tissue samples were collected for autoradiography and transmission electron microscopy (TEM) analysis. PET/CT imaging at 1-hr showed focal deposition of oil and nanoparticles in the tumor only after NE+ RFA but at 18-hrs, all animals had focal accumulation of oil and nanoparticles in the tumor region. Autoradiograph analysis demonstrated nanoparticle deposition in the tumor and in the ablated tissues adjacent to the tumor when NE was combined with ablation. TEM results showed the intracellular uptake of nanoparticles in tumor only after NE + IRE. Nanoparticles demonstrated a structural change, suggesting direct interaction, potentially leading to drug release, only after NE + LITT. The findings demonstrate that a combined NE and ablation treatment technique for liver tumors is feasible, resulting in deposition of nanoparticles in and around the tumor. Depending on the ablative energy applied, different effects are seen on nanoparticle localization and structure. These effects should be considered when designing nanoparticles for use in combination with ablation technologies.

Loss of CD73-mediated actin polymerization promotes endometrial tumor progression.

Ecto-5'-nucleotidase (CD73) is central to the generation of extracellular adenosine. Previous studies have highlighted a detrimental role for extracellular adenosine in cancer, as it dampens T cell-mediated immune responses. Here, we determined that, in contrast to other cancers, CD73 is markedly downregulated in poorly differentiated and advanced-stage endometrial carcinoma compared with levels in normal endometrium and low-grade tumors. In murine models, CD73 deficiency led to a loss of endometrial epithelial barrier function, and pharmacological CD73 inhibition increased in vitro migration and invasion of endometrial carcinoma cells. Given that CD73-generated adenosine is central to regulating tissue protection and physiology in normal tissues, we hypothesized that CD73-generated adenosine in endometrial carcinoma induces an innate reflex to protect epithelial integrity. CD73 associated with cell-cell contacts, filopodia, and membrane zippers, indicative of involvement in cell-cell adhesion and actin polymerization-dependent processes. We determined that CD73-generated adenosine induces cortical actin polymerization via adenosine A1 receptor (A1R) induction of a Rho GTPase CDC42-dependent conformational change of the actin-related proteins 2 and 3 (ARP2/3) actin polymerization complex member N-WASP. Cortical F-actin elevation increased membrane E-cadherin, ß-catenin, and Na(+)K(+) ATPase. Together, these findings reveal that CD73-generated adenosine promotes epithelial integrity and suggest why loss of CD73 in endometrial cancer allows for tumor progression. Moreover, our data indicate that the role of CD73 in cancer is more complex than previously described.

Coronary microvascular pericytes are the cellular target of sunitinib malate-induced cardiotoxicity.

Sunitinib malate is a multitargeted receptor tyrosine kinase inhibitor used in the treatment of human malignancies. A substantial number of sunitinib-treated patients develop cardiac dysfunction, but the mechanism of sunitinib-induced cardiotoxicity is poorly understood. We show that mice treated with sunitinib develop cardiac and coronary microvascular dysfunction and exhibit an impaired cardiac response to stress. The physiological changes caused by treatment with sunitinib are accompanied by a substantial depletion of coronary microvascular pericytes. Pericytes are a cell type that is dependent on intact platelet-derived growth factor receptor (PDGFR) signaling but whose role in the heart is poorly defined. Sunitinib-induced pericyte depletion and coronary microvascular dysfunction are recapitulated by CP-673451, a structurally distinct PDGFR inhibitor, confirming the role of PDGFR in pericyte survival. Thalidomide, an anticancer agent that is known to exert beneficial effects on pericyte survival and function, prevents sunitinib-induced pericyte cell death in vitro and prevents sunitinib-induced cardiotoxicity in vivo in a mouse model. Our findings suggest that pericytes are the primary cellular target of sunitinib-induced cardiotoxicity and reveal the pericyte as a cell type of concern in the regulation of coronary microvascular function. Furthermore, our data provide preliminary evidence that thalidomide may prevent cardiotoxicity in sunitinib-treated cancer patients.

Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells.

Phage display screening allows the study of functional protein-protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development.

Preparation, characterization, and cellular associations of silicon logic-embedded vectors.

Logic-embedded vectors (LEVs) have been introduced as a means to overcome sequential, biological barriers that prevent particle-based drug delivery systems from reaching their targets. In this chapter, we address the challenge of fabricating and optimizing LEVs to reach non-endosomal targets. We describe the general preparation, characterization, and cellular association of porous silicon-based LEVs. A specific example of LEV fabrication from start to finish, along with optimization and troubleshooting information, is presented to serve as a template for future designs.

Logic-embedded vectors for intracellular partitioning, endosomal escape, and exocytosis of nanoparticles.

A new generation of nanocarriers, logic-embedded vectors (LEVs), is endowed with the ability to localize components at multiple intracellular sites, thus creating an opportunity for synergistic control of redundant or dual-hit pathways. LEV encoding elements include size, shape, charge, and surface chemistry. In this study, LEVs consist of porous silicon nanocarriers, programmed for cellular uptake and trafficking along the endosomal pathway, and surface-tailored iron oxide nanoparticles, programmed for endosomal sorting and partitioning of particles into unique cellular locations. In the presence of persistent endosomal localization of silicon nanocarriers, amine-functionalized nanoparticles are sorted into multiple vesicular bodies that form novel membrane-bound compartments compatible with cellular secretion, while chitosan-coated nanoparticles escape from endosomes and enter the cytosol. Encapsulation within the porous silicon matrix protects these nanoparticle surface-tailored properties, and enhances endosomal escape of chitosan-coated nanoparticles. Thus, LEVs provide a mechanism for shielded transport of nanoparticles to the lesion, cellular manipulation at multiple levels, and a means for targeting both within and between cells.

Inhibition of mitochondrial metabolism by methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate induces apoptotic or autophagic cell death in chronic myeloid leukemia cells.

The initial success of the first synthetic bcr-abl kinase inhibitor imatinib has been dampened by the emergence of imatinib-resistant disease in blast crisis chronic myeloid leukemia. Here, we report that the novel triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate (CDDO-Me) potently induced cytotoxicity in imatinib-resistant KBM5 cells expressing the T315I mutation of bcr-abl (24-h EC50, 540 nmol/L). In long-term culture, CDDO-Me abrogated the growth of human parental KBM5 and KBM5-STI cells with 96-h IC50 of 205 and 221 nmol/L, respectively. In addition, CDDO-Me rapidly decreased the viability of murine lymphoid Ba/F3 cells expressing wild-type p210 as well as the imatinib-resistant E255K and T315I mutations of bcr-abl. The low-dose effects of CDDO-Me are associated with inhibition of mitochondrial oxygen consumption, whereas the cytotoxic effects appear to be mediated by a rapid and selective depletion of mitochondrial glutathione that accompanies the increased generation of reactive oxygen species and mitochondrial dysfunction. Interestingly, the mitochondriotoxic effects of CDDO-Me are followed by the rapid autophagocytosis of intracellular organelles or the externalization of phosphatidylserine in different cell types. We conclude that alterations in mitochondrial function by CDDO-Me can result in autophagy or apoptosis of chronic myeloid leukemia cells regardless of the mutational status of bcr-abl. CDDO-Me is in clinical trials and shows signs of clinical activity, with minimal side effects and complete lack of cardiotoxicity. Studies in leukemias are in preparation.

Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells.

The proteasome inhibitor bortezomib (formerly known as PS-341) recently received Food and Drug Administration approval for the treatment of multiple myeloma, and its activity is currently being evaluated in solid tumors. Bortezomib triggers apoptosis in pancreatic cancer cells, but the mechanisms involved have not been fully elucidated. Here, we show that pancreatic cancer cells exposed to bortezomib formed aggregates of ubiquitin-conjugated proteins ("aggresomes") in vitro and in vivo. Bortezomib-induced aggresome formation was determined to be cytoprotective and could be disrupted using histone deacetylase (HDAC) 6 small interfering RNA or chemical HDAC inhibitors, which resulted in endoplasmic reticulum stress and synergistic levels of apoptosis in vitro and in an orthotopic pancreatic cancer xenograft model in vivo. Interestingly, bortezomib did not induce aggresome formation in immortalized normal human pancreatic epithelial cells in vitro or in murine pancreatic epithelial cells in vivo. In addition, these cells did not undergo apoptosis following treatment with bortezomib, suberoylanilide hydroxamic acid, or the combination, showing tumor selectivity. Taken together, our study shows that inhibition of aggresome formation can strongly potentiate the efficacy of bortezomib and provides the foundation for clinical trials of bortezomib in combination with HDAC inhibitors for the treatment of pancreatic cancer.

Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL.

Previous studies showed that chronic lymphocytic leukemia (CLL) cells exhibit certain mitochondrial abnormalities including mtDNA mutations, increased superoxide generation, and aberrant mitochondrial biogenesis, which are associated with impaired apoptosis and reduced sensitivity to fludarabine. Here we report that CLL cells and multiple myeloma cells are highly sensitive to brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport currently being developed as a novel anticancer agent in a prodrug formulation. Of importance, brefeldin A effectively induced apoptosis in fludarabine-refractory CLL cells. Disruption of protein trafficking by brefeldin A caused the sequestration of the prosurvival factors APRIL and VEGF in the ER, leading to abnormal ER swelling and a decrease in VEGF secretion. Such ER stress and blockage of secretory protein traffic eventually resulted in Golgi collapse, activation of caspases, and cell death. Notably, the cellular sensitivity to this compound appeared to be independent of p53 status. Taken together, these findings suggest that malignant B cells may be highly dependent on ER-Golgi protein transport and that targeting this process may be a promising therapeutic strategy for B-cell malignancies, especially for those that respond poorly to conventional treatments.

Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinase and induces apoptosis via ER stress in human pancreatic cancer cells.

Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative that is a selective and potent inhibitor of the proteasome. We hypothesized that proteasome inhibition would lead to an accumulation of misfolded proteins in the cell resulting in endoplasmic reticulum (ER) stress. The ability of bortezomib to induce ER stress and the unfolded protein response was investigated in a human pancreatic cancer cell line, L3.6pl. Bortezomib increased expression of ER stress markers, CHOP and BiP, but inhibited PKR-like ER kinase and subsequent phosphorylation of eukaryotic initiation factor 2alpha (eif2alpha), both of which are key events in translational suppression. These effects resulted in an accumulation of ubiquitylated proteins leading to protein aggregation and proteotoxicity. Peptide inhibitor or small interfering RNA targeting ER-resident caspase-4 blocked DNA fragmentation, establishing a central role for caspase-4 in bortezomib-induced cell death. The translation inhibitor cycloheximide abrogated bortezomib-induced protein aggregation, caspase-4 processing, and all other characteristics of apoptosis. Because malignant cells have higher protein synthesis rates than normal cells, they may be more prone to protein aggregation and proteotoxicity and possess increased sensitivity to bortezomib-induced apoptosis. Taken together, the results show that bortezomib induces a unique type of ER stress compared with other ER stress agents characterized by an absence of eif2alpha phosphorylation, ubiquitylated protein accumulation, and proteotoxicity.

Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis.

Bortezomib (PS-341, Velcade) is a potent and selective inhibitor of the proteasome that is currently under investigation for the treatment of solid malignancies. We have shown previously that bortezomib has activity in pancreatic cancer models and that the drug induces endoplasmic reticulum (ER) stress but also suppresses the unfolded protein response (UPR). Because the UPR is an important cytoprotective mechanism, we hypothesized that bortezomib would sensitize pancreatic cancer cells to ER stress-mediated apoptosis. Here, we show that bortezomib promotes apoptosis triggered by classic ER stress inducers (tunicamycin and thapsigargin) via a c-Jun NH(2)-terminal kinase (JNK)-dependent mechanism. We also show that cisplatin stimulates ER stress and interacts with bortezomib to increase ER dilation, intracellular Ca(2+) levels, and cell death. Importantly, combined therapy with bortezomib plus cisplatin induced JNK activation and apoptosis in orthotopic pancreatic tumors resulting in a reduction in tumor burden. Taken together, our data establish that bortezomib sensitizes pancreatic cancer cells to ER stress-induced apoptosis and show that bortezomib strongly enhances the anticancer activity of cisplatin.