Onconase responsive genes in human mesothelioma cells: implications for an RNA damaging therapeutic agent

<p>Abstract</p> <p>Background</p> <p>Onconase represents a new class of RNA-damaging drugs. Mechanistically, Onconase is thought to internalize, where it degrades intracellular RNAs such as tRNA and double-stranded RNA, and thereby suppresses protein synthesis. However, there may be additional or alternative mechanism(s) of action.</p> <p>Methods</p> <p>In this study, microarray analysis was used to compare gene expression profiles in untreated human malignant mesothelioma (MM) cell lines and cells exposed to 5 μg/ml Onconase for 24 h. A total of 155 genes were found to be regulated by Onconase that were common to both epithelial and biphasic MM cell lines. Some of these genes are known to significantly affect apoptosis (IL-24, TNFAIP3), transcription (ATF3, DDIT3, MAFF, HDAC9, SNAPC1) or inflammation and the immune response (IL-6, COX-2). RT-PCR analysis of selected up- or down-regulated genes treated with varying doses and times of Onconase generally confirmed the expression array findings in four MM cell lines.</p> <p>Results</p> <p>Onconase treatment consistently resulted in up-regulation of IL-24, previously shown to have tumor suppressive activity, as well as ATF3 and IL-6. Induction of ATF3 and the pro-apoptotic factor IL-24 by Onconase was highest in the two most responsive MM cell lines, as defined by DNA fragmentation analysis. In addition to apoptosis, gene ontology analysis indicated that pathways impacted by Onconase include MAPK signaling, cytokine-cytokine-receptor interactions, and Jak-STAT signaling.</p> <p>Conclusions</p> <p>These results provide a broad picture of gene activity after treatment with a drug that targets small non-coding RNAs and contribute to our overall understanding of MM cell response to Onconase as a therapeutic strategy. The findings provide insights regarding mechanisms that may contribute to the efficacy of this novel drug in clinical trials of MM patients who have failed first line chemotherapy or radiation treatment.</p

Rescue of replication failure by Fanconi anaemia proteins

Chromosomal aberrations are often associated with incomplete genome duplication, for instance at common fragile sites, or as a consequence of chemical alterations in the DNA template that block replication forks. Studies of the cancer-prone disease Fanconi anaemia (FA) have provided important insights into the resolution of replication problems. The repair of interstrand DNA crosslinks induced by chemotherapy drugs is coupled with DNA replication and controlled by FA proteins. We discuss here the recent discovery of new FA-associated proteins and the development of new tractable repair systems that have dramatically improved our understanding of crosslink repair. We focus also on how FA proteins protect against replication failure in the context of fragile sites and on the identification of reactive metabolites that account for the development of Fanconi anaemia symptoms

Synergism Between PDE4 and PI3Kδ Inhibitors in DLBCL: Improved Clinical Activity with the Potential for Lower Toxicity

Abstract Aberrant activation of the B cell receptor (BCR) is a hallmark of mature B-cell tumors. A better understanding of this process will spearhead effective clinical translation. The initiation and amplification of BCR signaling are well-defined events, and the successful deployment of BTK and PI3Kδ inhibitors in the clinic capitalizes on this knowledge. Conversely, the intricacies of the termination of BCR signals are less well-understood, and to date no rational therapeutic approach has been developed that exploit this aspect of the oncogenic BCR. Cyclic-AMP (cAMP) is a second messenger with marked growth suppression properties towards immune cells, including neoplastic mature B lymphocytes. In earlier work, we showed that inhibition of phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP, downmodulates the activity of classical effectors of BCR signals, including SYK and PI3K. Herein, we attempted to gain further mechanistic understanding on how cAMP suppresses the proximal BCR activity, and built on this information to pre-clinically test therapeutic strategies that simultaneously attack the BCR at its amplification and termination points. Using diffuse large B cell lymphoma (DLBCL) as a model, we focused our attention on the interplay between cAMP and LCK, as we unexpectedly found that this cAMP-regulated canonical T-cell kinase is also widely expressed in DLBCL. Working with LCK-positive PDE4-low/null DLBCL cell lines, we found a marked increase in the phosphorylation of the inhibitory Y505 of LCK following elevation of intra-cellular cAMP. Next, we showed that ectopic expression of wild-type (WT) PDE4B, but not of a phosphodiesterase-inactive (PI) mutant, abrogated the cAMP-mediated, CSK-dependent, phosphorylation of LCK. Active LCK can phosphorylate PI3K's p85 regulatory subunit, thus freeing the catalytic domain from its structural constraints to promote lipid kinase activity. Thus, we tested whether the cAMP-mediated inhibition of LCK, by suppressing p85 phosphorylation, down-modulated PI3K activity. In LCK-positive PDE4B-null DLBCL, we showed that cAMP readily decreased the phosphorylation of p85 that followed BCR engagement; using the WT and PI PDE4B genetic models, we demonstrated that PDE4B expression abrogated cAMP effects and led to sustained PI3K activity following BCR engagement. These data suggested that inhibition of PDE4, by unleashing the negative effects of cAMP on LCK/p85, could accelerate the termination of PI3K activation that follows BCR engagement. If this hypothesis was correct, then the combination of PI3K and PDE4 inhibitors by attacking the BCR at its amplification and termination points, respectively, may synergistically suppress the growth of DLBCL. In in vitro studies with multiple DLBCL cell lines (WSU-NHL, OCI-Ly7, OCI-Ly18, OCI-Ly3, HBL-1, and OCI-Ly10) we showed that the combination of the FDA-approved PDE4 inhibitor roflumilast with idelalisib synergistically suppresses DLBCL growth (combination index &lt; 1). This synergism was associated with a significant suppression of PI3K and AKT activities (p&lt;0.05, cells treated with the drug combination vs. single agents). We expanded this observation to an in vivo xenograft model of human DLBCL, and showed that mice treated with roflumilast and idelalisib had a significantly smaller tumor burden than those receiving single agents (p&lt; 0.01, two cohorts, n=47 mice). We also found a greater suppression of PI3K activity in the xenografts from mice treated with the combination of PDE4 and PI3Kδ inhibitors (p&lt; 0.0001), as well as increased apoptosis. Together, these data further delineated how cAMP suppresses the BCR and showed that the rational combination PDE4 and PI3Kδ inhibitors synergistically suppresses DLBCL growth. These results are particularly important given the recent evidence of inflammatory/immune toxicity associated with the use of idelalisib, which we propose could be countered by the well-established anti-inflammatory properties of PDE4 inhibitors. Thus, we hypothesize that combining PDE4 and PI3Kδ inhibitors will enhance anti-lymphoma activity while decreasing clinical toxicity. This concept is ripe for clinical testing as we have recently completed a phase Ib trial showing that roflumilast is safe and active in patients with advanced B cell malignancies. Disclosures No relevant conflicts of interest to declare. </jats:sec

A Phosphodiesterase 4B-Mediated Interplay Between Tumor Cells and the Microenvironment Regulates Angiogenesis in B Cell Lymphoma

Abstract Diffuse large B cell lymphoma (DLBCL) is a common and heterogeneous tumor. Extensive genetic examinations of these malignancies were performed in the past decade, but this knowledge has yet to be translated into rationally-designed treatment strategies that effectively change its cure rate. Recognizing and acquiring basic biology data in specific priority areas may accelerate clinical translation in DLBCL. One such knowledge gap concerns the interplay between lymphoma cells, the microenvironment and angiogenesis. This is particularly important because high circulating levels of vascular endothelial growth factor (VEGF) and elevated microvessel density (MVD) are associated with poor outcome in DLBCL, while clinical trials that tested classical anti-VEGFA agents in this setting were negative and plagued by serious adverse events. Cyclic-AMP (cAMP) is a pervasive second messenger that in immune cells exerts primarily negative effects, including suppression of proximal B or T cell receptor signaling and induction of apoptosis. In immune cells, cAMP signaling is terminated by phosphodiesterase 4 (PDE4). Earlier, we identified PDE4B in an outcome prediction signature of DLBCL and showed subsequently that its inhibition had anti-lymphoma properties. cAMP activity is also highly contextualized and it was recently suggested to attenuate vessel development in non-neoplastic cell models. Thus, we speculated that high PDE4B expression/activity, by abrogating cAMP signaling, could modulate angiogenesis in DLBCL. To examine this idea, we first used a panel of DLBCL cell lines and found that cAMP suppressed VEGF expression (mRNA) and secretion (protein) in PDE4B-low but not in PDE4B-high DLBCLs. In human umbilical vein endothelial cell (HUVEC) tube formation assays, we noted that conditioned media from PDE4B-high DLBCLs were significantly more angiogenic than those from PDE4B-low models. To isolate the role of PDE4B in this process, we used genetic and pharmacological models. Stable ectopic expression of PDE4B blocked the anti-angiogenic properties of cAMP, whereas a siRNA-mediated PDE4B knockdown, or exposure to the FDA-approved PDE4 inhibitor Roflumilast, suppressed VEGF levels and activity. Mechanistically, we demonstrated that cAMP, in a PDE4B-dependent manner, suppresses PI3K and AKT activities to impose its anti-angiogenic properties. Thus, ectopic expression of a constitutively active AKT gene in PDE4-low DLBCL cell lines abrogated cAMP effects in a manner similar to PDE4B reconstitution, indicating that PI3K/AKT are key mediators of the cAMP/PDE4 effects on angiogenesis. To expand these observations to more elaborate models, we created a composite mouse where c-Myc-driven lymphomas develop in Pde4b null or wild-typebackgrounds. Remarkably, primary lymphomas from Eµ-Myc;Pde4b-/- mice displayed significantly lower MVD (quantified by immunohistochemistry - IHC - with anti-CD34 staining) than the lymphomas that developed in Eµ-Myc;Pde4b+/+ mice (n= 19, p&lt;0.001). Validating our in vitro data, the primary B cell lymphomas originating in the Pde4b-/- background displayed lower PI3K activity, AKT phosphorylation (n=13, p&lt;0.01) and VEGF levels (determined by IHC, n=18, p=0.01). Next, we tested the hypothesis that pharmacological inhibition of PDE4 in vivo could effectively suppress lymphoma angiogenesis. To that end, we used adoptive transfer to generate multiple independent cohorts of Eµ-Myc-driven lymphoma-bearing mice (n=68), which were randomized to receive vehicle or Roflumilast (5mg/kd/day gavage). B cell lymphomas from Roflumilast-treated mice showed a marked suppression of angiogenesis (p=0.01, for MVD of Roflumilast vs. vehicle groups) and significant decrease in PI3K/AKT activity (p=0.003), which were accompanied by lower serum levels of VEGF (p=0.005). In addition, in comparison to their vehicle-treated isogenic controls, mice that received Roflumilast displayed a smaller tumor burden (p&lt;0.0001) and improved survival (p=0.01). Lastly, we examined a series of primary human DLBCLs (n=28) and confirmed a significant direct correlation between PDE4B levels and microvessel density in these specimens (r=0.43, p=0.02). Together, these data uncover a novel signaling cross-talk between lymphoma cells and the microenvironment that regulates angiogenesis in vivo. Our findings point to PDE4 as actionable proangiogenic factor in B cell lymphomas Disclosures No relevant conflicts of interest to declare. </jats:sec

Synergistic Targeting of the Regulatory and Catalytic Subunits of PI3Kδ in Mature B-cell Malignancies

Abstract Purpose: Aberrant activation of the B-cell receptor (BCR) is implicated in the pathogenesis of mature B-cell tumors, a concept validated in part by the clinical success of inhibitors of the BCR-related kinases BTK (Bruton's tyrosine kinase) and PI3Kδ. These inhibitors have limitations, including the paucity of complete responses, acquired resistance, and toxicity. Here, we examined the mechanism by which the cyclic-AMP/PDE4 signaling axis suppresses PI3K, toward identifying a novel mechanism-based combinatorial strategy to attack BCR-dependency in mature B-cell malignancies. Experimental Design: We used in vitro and in vivo diffuse large B-cell lymphoma (DLBCL) cell lines and primary chronic lymphocytic leukemia (CLL) samples to preclinically evaluate the effects of the combination of the FDA-approved phosphodiesterase 4 (PDE4) inhibitor roflumilast and idelalisib on cell survival and tumor growth. Genetic models of gain- and loss-of-function were used to map multiple signaling intermediaries downstream of the BCR. Results: Roflumilast elevates the intracellular levels of cyclic-AMP and synergizes with idelalisib in suppressing tumor growth and PI3K activity. Mechanistically, we show that roflumilast suppresses PI3K by inhibiting BCR-mediated activation of the P85 regulatory subunit, distinguishing itself from idelalisib, an ATP-competitive inhibitor of the catalytic P110 subunit. Using genetic models, we linked the PDE4-regulated modulation of P85 activation to the oncogenic kinase SYK. Conclusions: These data demonstrate that roflumilast and idelalisib suppress PI3K by distinct mechanisms, explaining the basis for their synergism, and suggest that the repurposing of PDE4 inhibitors to treat BCR-dependent malignancies is warranted. Clin Cancer Res; 24(5); 1103–13. ©2017 AACR.</jats:p

Suppl Table S2 from Safety and Pharmacodynamics of the PDE4 Inhibitor Roflumilast in Advanced B-cell Malignancies

Steady-state trough levels of roflumilast and its active metabolite N-oxide.</p

Supplementary Figure Legend from Safety and Pharmacodynamics of the PDE4 Inhibitor Roflumilast in Advanced B-cell Malignancies

Supplementary Figure Legend</p