Single-agent lenalidomide in relapsed/refractory mantle cell lymphoma: results from a UK phase II study suggest activity and possible gender differences.

We present data from a phase II study investigating a novel treatment strategy for relapsed/refractory mantle cell lymphoma (MCL). Twenty-six patients received lenalidomide 25 mg/d (days 1-21 of a 28-d cycle) for up to 6 cycles followed by low-dose maintenance lenalidomide (15 mg) in responding patients. Eight patients achieved complete or partial response to give an overall response rate of 31% with median response duration of 22·2 months [95% confidence interval (CI) 0·0-53·6] and median progression-free survival (PFS) of 3·9 months (95% CI 0·0-11·1). An additional six patients (23%) achieved stable disease. Eleven patients received maintenance with median PFS of 14·6 months (95% CI 7·3-21·9). Correlative studies showed that peripheral T and Natural Killer (NK) cells increased in responding patients by 40-60% over the first 6 cycles with an initial dip in NK cells suggestive of tumour infiltration. Peripheral regulatory T cells were increased in MCL patients (P = 0·001) and expanded further following lenalidomide. Sequential plasma analysis showed increased IL12 p40 and IL7 alongside decreased MMP9, IL10, and adiponectin. Finally, a significant correlation (P = 0·02) between gender and response suggested that female MCL patients were more sensitive to lenalidomide than males. In summary, we confirm the activity, safety and immunomodulatory properties of lenalidomide in MCL and highlight its potential as a low-dose maintenance agent.

Increased cellular accumulation and distribution of amrubicin contribute to its activity in anthracycline-resistant cancer cells.

PURPOSE: Multi-drug resistance and cumulative cardiotoxicity are major limitations for the clinical use of anthracyclines. Here, we evaluated and compared the cross-resistance of amrubicin, a third-generation synthetic anthracycline and potent topoisomerase (topo)-II inhibitor with little or no observed cardiotoxicity to other anthracyclines and the topo-II inhibitor etoposide in drug-resistant tumor models in order to elucidate its potential mechanisms of action. METHODS: Amrubicin activity was assessed in multi-drug-resistant cell lines and human tumor explants using cytotoxicity assays, confocal microscopy, fluorescence time-lapse imaging, flow cytometry, immunoblotting, and gene expression profiling techniques. RESULTS: We demonstrate that both doxorubicin-resistant tumor cell lines and several drug-resistant human ovarian and breast tumor explants retain sensitivity to amrubicin. In addition, we observed similar levels of amrubicin uptake and accumulation in doxorubicin-sensitive versus doxorubicin-resistant cell lines. Although amrubicin is a weak P-glycoprotein substrate, transport and retention of amrubicin were not solely modulated by P-glycoprotein in the resistant cell lines overexpressing drug efflux pumps. The cellular retention of amrubicin is likely to be a result of rapid influx due to its high intrinsic permeability and lipophilic properties, and this may explain why amrubicin overcomes pleiotropic drug resistance. Consistent with drug accumulation studies, amrubicin induced DNA damage, G(2)-M cell cycle arrest, and apoptosis in both doxorubicin-sensitive and doxorubicin-resistant lines. Using gene expression profiling studies, several classes of genes were significantly and uniquely regulated following amrubicin, but not doxorubicin or etoposide, treatment. CONCLUSIONS: Amrubicin appears to have a distinct mode of action that overcomes typical anthracycline resistance mechanisms. Therefore, amrubicin may be useful in the treatment of anthracycline-refractory or anthracycline-resistant tumors.

The class-I HDAC inhibitor MGCD0103 induces apoptosis in Hodgkin lymphoma cell lines and synergizes with proteasome inhibitors by an HDAC6-independent mechanism.

Inhibition of histone deacetylase 6 (HDAC6)-dependent aggresome function by pan HDAC inhibitors was recently reported to be a key mechanism underlying the synergistic activity between proteasome inhibitors and HDAC inhibitors in a variety of tumour types. Because these combinations induce significant thrombocytopenia in vivo, we examined whether less toxic, isotype-selective HDAC inhibitors may still synergize with proteasome inhibitors, and if so, by what mechanisms. Here, we showed that the class I HDAC inhibitor, MGCD0103, has a potent antiproliferative activity in Hodgkin lymphoma (HL) cell lines. Furthermore, MGCD0103 induced tumour necrosis factor α (TNF-α) expression and secretion, which was associated with nuclear factor (NF)-κB activation. Selective inhibition of TNF-α expression by short interfering mRNA, or inhibition of MGCD0103-induced NF-kB activation by proteasome inhibitors enhanced MGCD0103-induced cell death. Thus, our results demonstrate that MGCD0103 may synergize with proteasome inhibitors by HDAC6-independent mechanisms, providing mechanistic rationale for exploring this potentially less toxic combination for the treatment of lymphoma.

Peptide modulators of Src activity in G1 regulate entry into S phase and proliferation of NIH 3T3 cells.

Cascades of kinases and phosphatases are regulated by selective protein-protein interactions that are essential for signal transduction. Peptide modulators of these interactions have been used to dissect the function of individual components of the signaling cascade, without relying on either the over- or underexpression of proteins. Previously, we identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src tyrosine kinases. Here, we utilized cell-permeable peptides that selectively disrupt or enhance the interaction of RACK1 and Src to further examine the function of RACK1. Our results provide direct physiologic evidence that RACK1 regulates growth of NIH3T3 cells by suppressing the activity of Src and other cell cycle regulators in G1, and delaying entry into S phase. They also demonstrate the potential for using peptide modulators of Src activity as a tool for regulating cell growth, and for designing new strategies for cancer therapy that target specific protein-protein interactions.

RACK1 regulates G1/S progression by suppressing Src kinase activity.

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.

RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells.

Cancer cells are capable of serum- and anchorage-independent growth, and focus formation on monolayers of normal cells. Previously, we showed that RACK1 inhibits c-Src kinase activity and NIH3T3 cell growth. Here, we show that RACK1 partially inhibits v-Src kinase activity, and the serum- and anchorage-independent growth of v-Src transformed cells, but has no effect on focus formation. RACK1-overexpressing v-Src cells show disassembly of podosomes, which are actin-rich structures that are distinctive to fully transformed cells. Together, our results demonstrate that RACK1 overexpression in v-Src cells partially reverses the transformed phenotype of the cells. Our results identify an endogenous inhibitor of the oncogenic Src tyrosine kinase and of cell transformation.

Regulation of interleukin receptor-associated kinase (IRAK) phosphorylation and signaling by iota protein kinase C.

We have previously shown that the activity of the interleukin-1 (IL-1) receptor-associated kinase (IRAK) is required for nerve growth factor (NGF)-induced activation of NF-kappaB and cell survival ((2002) J. Biol. Chem. 277, 28010-28018). Herein we demonstrate that NGF induces co-association of IRAK with atypical protein kinase C iota (PKC) and that the iota PKC.IRAK complex is recruited to the p75 neurotrophin receptor. Recruitment of IRAK to the receptor was dependent upon the activity of the iota PKC. Moreover, transfection of kinase-dead iota PKC blocked both NGF- and IL-1-induced IRAK activation and the activity of NF-kappaB. Hence, iota PKC lies upstream of IRAK in the kappaB pathway. Examining the primary structure of IRAK, we identified three putative PKC phosphorylation sites; iota PKC selectively phosphorylated peptide 1 (RTAS) within the death domain domain at Thr66, which is highly conserved among all IRAK family members. Mutation of Thr66 to Ala impaired the autokinase activity of IRAK and reduced its association with iota PKC but not TRAF6, resulting in impaired NGF- as well as IL-1-induced NF-kappaB activation. These findings provide insight into the underlying mechanism whereby IRAK regulates the kappaB pathway and reveal that IRAK is a substrate of iota PKC.

Dual role for p75(NTR) signaling in survival and cell death: can intracellular mediators provide an explanation?

Several recent reports support a dual role of p75(NTR) in cell death, as well as survival, depending on the physiological or developmental stage of the cells. Coexpression of the TrkA receptor with p75(NTR) further enhances the complexity of nerve growth factor (NGF) signaling. Recent identification of serine/threonine kinases that interact with the p75(NTR) provides an explanation for the lack of an apparent kinase domain needed for signaling. In this report, we review the possible roles of the intracellular proteins that directly interact with the p75(NTR), atypical protein kinase C (PKC) binding protein, p62 and second messengers in the functional antagonism exhibited by TrkA and p75(NTR) with an emphasis on the nuclear factor-kappa B activation pathway.

Identification of interleukin 1 receptor-associated kinase as a conserved component in the p75-neurotrophin receptor activation of nuclear factor-kappa B.

The neurotrophin nerve growth factor (NGF) supports neuronal survival by activating the transcription factor nuclear factor-kappaB (NF-kappaB). We report here, for the first time, the identification of p75-associated kinase that mediates NGF-driven NF-kappaB activation. Using co-immunoprecipitation, we demonstrate an NGF-dependent association of interleukin 1 receptor-associated kinase (IRAK) with the p75 neurotrophin receptor in PC12 cells. Our results reveal that IRAK is recruited to the p75-NGF receptor leading to formation of a complex between IRAK, atypical protein kinase C interacting protein, p62, and TRAF6. Activation of NF-kappaB occurs predominantly through the p75 receptor, and TrkA activity suppresses NF-kappaB activation and retards IkappaBbeta degradation. In addition, we observe a requirement for the kinase activity of IRAK in mediating NGF-induced NF-kappaB activation, recruitment of the adapter protein p62 to the p75 receptor, and cell survival. Moreover, p75-IRAK-mediated kappaB activation and the recruitment of IKKbeta, but not IKKalpha, to the receptor require p62. Altogether, our data provide novel information regarding the proximal components involved in p75 receptor signaling and underscore the importance of the atypical PKC interacting protein p62 in this process.

Nerve growth factor activates kinases that phosphorylate atypical protein kinase C.

Activation of atypical protein kinase C by nerve growth factor (NGF) involves phosphorylation. In order to identify kinases that regulate atypical PKC (aPKC), we surveyed PC12 cell lysates for protein kinases that are activated by NGF and which could phosphorylate aPKC. Employing an in-gel kinase assay where aPKC-zeta was copolymerized within the gel matrix as a substrate, three kinases, pp175, pp87 and pp60, were identified as enzymes that phosphorylated aPKC. Phosphorylation of aPKC by these three kinases coincided with NGF-induced activation of the enzyme. Each kinase possessed a unique subcellular distribution pattern and could be activated by either ceramide or H(2)0(2), second messengers that mimic NGF signaling events. Upstream, pp175 and pp60 lie in a ras pathway, whereas pp87 lies in a pathway dependent upon src. Altogether, these findings reveal that the aPKCs are subject to regulation by a novel group of kinases.