Evidence of mTOR Activation by an AKT-Independent Mechanism Provides Support for the Combined Treatment of PTEN-Deficient Prostate Tumors with mTOR and AKT Inhibitors

AbstractActivation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone

IAP inhibitors enhance co-stimulation to promote tumor immunity

The inhibitor of apoptosis proteins (IAPs) have recently been shown to modulate nuclear factor κB (NF-κB) signaling downstream of tumor necrosis factor (TNF) family receptors, positioning them as essential survival factors in several cancer cell lines, as indicated by the cytotoxic activity of several novel small molecule IAP antagonists. In addition to roles in cancer, increasing evidence suggests that IAPs have an important function in immunity; however, the impact of IAP antagonists on antitumor immune responses is unknown. In this study, we examine the consequences of IAP antagonism on T cell function in vitro and in the context of a tumor vaccine in vivo. We find that IAP antagonists can augment human and mouse T cell responses to physiologically relevant stimuli. The activity of IAP antagonists depends on the activation of NF-κB2 signaling, a mechanism paralleling that responsible for the cytotoxic activity in cancer cells. We further show that IAP antagonists can augment both prophylactic and therapeutic antitumor vaccines in vivo. These findings indicate an important role for the IAPs in regulating T cell–dependent responses and suggest that targeting IAPs using small molecule antagonists may be a strategy for developing novel immunomodulating therapies against cancer

Development of Peptidomimetics Targeting IAPs

Inhibitor of apoptosis proteins (IAPs) such as XIAP subvert apoptosis by binding and inhibiting caspases. Because occupation of the XIAP BIR3 peptide binding pocket by Smac abolishes the XIAP–caspase 9 interaction, it is a proapoptotic event of great therapeutic interest. An assay for pocket binding was developed based on the displacement of Smac 7-mer from BIR3. Through the physical and biochemical analysis of a variety of peptides, we have determined the minimum sequence required for inhibition of the Smac–BIR3 interaction and detailed the dimensions and topology of the BIR3 peptide binding pocket. This work describes the structure–activity relationship (SAR) for peptide inhibitors of Smac-IAP binding

Common themes in assembly and function of eukaryotic transcription complexes.

Eukaryotes contain three distinct RNA polymerase enzymes, each responsible for the transcription of a subclass of nuclear genes. Despite this division of labor, each RNA polymerase system follows a common blueprint to execute the loading of the polymerase onto the relevant promoter region. The RNA polymerase II system appears unique in that after RNA polymerase II has loaded onto the DNA, two auxiliary factors, TFIIE and TFIIH, are necessary for its escape from the promoter region. The complexity of the RNA polymerase II initiation pathway provides a multitude of potential targets for transcriptional activators. Tight control over transcription initiation levels is afforded by multiple cofactors that both enhance and repress

Recycling of the general transcription factors during RNA polymerase II transcription.

We have analyzed the fate of the RNA polymerase II (RNAPII) general transcription factors during the transition from initiation to elongation using multiple approaches. We demonstrate that all of the basal factors coexist in mature initiation complexes but that following nucleotide addition, this complex becomes disrupted. During this transition, TFIID remains promoter-bound whereas TFIIB, TFIIE, TFIIF, and TFIIH are released. Upon release, TFIIB reassociates with TFIID, reforming the RNAPII docking site, the DB complex. TFIIE is released before formation of the tenth phosphodiester bond. This precedes TFIIH release, which occurrs after the transcription complex reaches +30. TFIIF is unique in that it is the only basal factor detected in the RNAPII elongation complex. Following its release from the initiation complex, TFIIF has the ability to reassociate with a stalled RNAPII

A small molecule IAP inhibitor sensitises medulloblastoma cells to radiation therapy and cytotoxic chemotherapy in vitro and in vivo

Novel strategies for treating medulloblastoma are urgently needed. Despite intensive treatment one quarter of patients ultimately succumb to their disease. Radiation therapy is a critical component of current treatment strategies but often results in devastating long-term effects, particularly in younger patients. Strategies to sensitise medulloblastoma cells to the effects of radiation therapy have been proposed as a mechanism to both decrease toxicity and enhance efficacy. The Inhibitor of Apoptosis Proteins (IAPs) represent the last molecular barrier to programmed cell death. They have been shown to be over-expressed in medulloblastoma and to correlate with poor patient outcomes. We hypothesized that targeting the IAPs in conjunction with conventional cytotoxic therapies would overcome treatment resistance, increase levels of apoptosis and significantly enhance their anti-tumor activity. We have previously shown that small molecule IAP inhibitor LBW242 penetrates the blood brain barrier and has a significant anti-tumour effect in intracerebral tumours such as high grade gliomas. We therefore sought to test LBW242 in medulloblastoma in combination with the standard-of-care therapies of irradiation and cytotoxic chemotherapy. In vitro experiments demonstrated that administration of LBW242 in combination with radiotherapy and chemotherapy led to both caspase 8 and 9 activation resulting in direct activation of both the intrinsic and extrinsic apoptotic pathways. Annexin staining showed that this ultimately led to medulloblastoma cell death and clonogenic assays showed that the combination therapy led to a synergistic anti-medulloblastoma effect in multiple cell lines. Athymic mice bearing established human medulloblastoma tumor xenografts treated with LBW242 plus cisplatinum demonstrated a profound and synergistic suppression of tumor growth in the in vivo setting. Taken together these experiments show that the pro-apoptotic and anti-tumour effects of radiotherapy and chemotherapy can be enhanced by the addition of a small molecule, orally bioavailable IAP inhibitor. These results are readily translatable to clinical trial, and offer the potential for improved treatment outcomes for medulloblastoma patients