Nanoencapsulation of MDM2 Inhibitor RG7388 and Class-I HDAC Inhibitor Entinostat Enhances their Therapeutic Potential Through Synergistic Antitumor Effects and Reduction of Systemic Toxicity.

Inhibitors of the p53-MDM2 interaction such as RG7388 have been developed to exploit latent tumor suppressive properties in p53 in 50% of tumors in which p53 is wild-type. However, these agents for the most part activate cell cycle arrest rather than death, and high doses in patients elicit on-target dose-limiting neutropenia. Recent work from our group indicates that combination of p53-MDM2 inhibitors with the class-I HDAC inhibitor Entinostat (which itself has dose-limiting toxicity issues) has the potential to significantly augment cell death in p53 wild-type colorectal cancer cells. We investigated whether coencapsulation of RG7388 and Entinostat within polymeric nanoparticles (NPs) could overcome efficacy and toxicity limitations of this drug combination. Combinations of RG7388 and Entinostat across a range of different molar ratios resulted in synergistic increases in cell death when delivered in both free drug and nanoencapsulated formats in all colorectal cell lines tested. Importantly, we also explored the in vivo impact of the drug combination on murine blood leukocytes, showing that the leukopenia induced by the free drugs could be significantly mitigated by nanoencapsulation. Taken together, this study demonstrates that formulating these agents within a single nanoparticle delivery platform may provide clinical utility beyond use as nonencapsulated agents.

Functional Genomic Identification of Predictors of Sensitivity and Mechanisms of Resistance to Multivalent Second-Generation TRAIL-R2 Agonists.

Multivalent second-generation TRAIL-R2 agonists are currently in late preclinical development and early clinical trials. Herein, we use a representative second-generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models, whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to suppress MEDI3039-induced cell death. High-throughput drug screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics analysis revealed striking association between MEDI3039 sensitivity and expression of core components of the extrinsic apoptotic pathway, most notably its main apoptotic effector caspase-8 in solid tumor cell lines. Further analyses of colorectal cell lines and patient-derived xenografts identified caspase-8 expression ratio to its endogenous regulator FLIP(L) as predictive of sensitivity to MEDI3039 in several major solid tumor types and a further subset indicated by caspase-8:MCL-1 ratio. Subsequent MEDI3039 combination screening of TRAIL-R2, caspase-8, FADD, and BID knockout models with 60 compounds with varying mechanisms of action identified two inhibitor of apoptosis proteins (IAP) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BID-deleted models. In summary, we identify the ratios of caspase-8:FLIP(L) and caspase-8:MCL-1 as potential predictive biomarkers for second-generation TRAIL-R2 agonists and loss of key effectors such as FADD and caspase-8 as likely drivers of clinical resistance in solid tumors.

Clinical Positioning of the IAP Antagonist Tolinapant (ASTX660) in Colorectal Cancer.

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation, and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer. We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in patients with microsatellite stable (MSS) stage III colorectal cancer treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy, suggesting their involvement in promoting chemoresistance. A novel IAP antagonist tolinapant (ASTX660) potently and rapidly downregulated cIAP1 in colorectal cancer models, demonstrating its robust on-target efficacy. In cells co-cultured with TNFα to mimic an inflammatory tumor microenvironment, tolinapant induced caspase-8-dependent apoptosis in colorectal cancer cell line models; however, the extent of apoptosis was limited because of inhibition by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinapant-induced apoptosis was augmented by FOLFOX in human colorectal cancer and murine organoid models in vitro and in vivo, due (at least in part) to FOLFOX-induced downregulation of class I histone deacetylases (HDAC), leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, the effects of FOLFOX could be phenocopied using the clinically relevant class I HDAC inhibitor, entinostat, which also induced acetylation of Ku70 and FLIP downregulation. Further analyses revealed that caspase-8 knockout RIPK3-positive colorectal cancer models were sensitive to tolinapant-induced necroptosis, an effect that could be exploited in caspase-8-proficient models using the clinically relevant caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX in poor prognosis MSS colorectal cancer with elevated cIAP1/2 expression.

Dying to Survive-The p53 Paradox.

The p53 tumour suppressor is best known for its canonical role as "guardian of the genome", activating cell cycle arrest and DNA repair in response to DNA damage which, if irreparable or sustained, triggers activation of cell death. However, despite an enormous amount of work identifying the breadth of the gene regulatory networks activated directly and indirectly in response to p53 activation, how p53 activation results in different cell fates in response to different stress signals in homeostasis and in response to p53 activating anti-cancer treatments remains relatively poorly understood. This is likely due to the complex interaction between cell death mechanisms in which p53 has been activated, their neighbouring stressed or unstressed cells and the local stromal and immune microenvironment in which they reside. In this review, we evaluate our understanding of the burgeoning number of cell death pathways affected by p53 activation and how these may paradoxically suppress cell death to ensure tissue integrity and organismal survival. We also discuss how these functions may be advantageous to tumours that maintain wild-type p53, the understanding of which may provide novel opportunity to enhance treatment efficacy.

The pseudo-caspase FLIP(L) regulates cell fate following p53 activation.

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Loss of nitric oxide-mediated inhibition of purine neurotransmitter release in the colon in the absence of interstitial cells of Cajal.

Regulation of colonic motility depends on the integrity of enteric inhibitory neurotransmission mediated by nitric oxide (NO), purine neurotransmitters, and neuropeptides. Intramuscular interstitial cells of Cajal (ICC-IM) and platelet-derived growth factor receptor-α-positive (PDGFRα+) cells are involved in generating responses to NO and purine neurotransmitters, respectively. Previous studies have suggested a decreased nitrergic and increased purinergic neurotransmission in KitW/KitW-v (W/Wv ) mice that display lesions in ICC-IM along the gastrointestinal tract. However, contributions of NO to these phenotypes have not been evaluated. We used small-chamber superfusion assays and HPLC to measure the spontaneous and electrical field stimulation (EFS)-evoked release of nicotinamide adenine dinucleotide (NAD+)/ADP-ribose, uridine adenosine tetraphosphate (Up4A), adenosine 5'-triphosphate (ATP), and metabolites from the tunica muscularis of human, monkey, and murine colons and circular muscle of monkey colon, and we tested drugs that modulate NO levels or blocked NO receptors. NO inhibited EFS-evoked release of purines in the colon via presynaptic neuromodulation. Colons from W/Wv, Nos1-/- , and Prkg1-/- mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/Wv mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. W/Wv mouse colons demonstrated reduced Nos1 expression and reduced NO release. Our results suggest that enhanced purinergic neurotransmission may compensate for the loss of nitrergic neurotransmission in muscles with partial loss of ICC. The interactions between nitrergic and purinergic neurotransmission in the colon provide novel insight into the role of neurotransmitters and effector cells in the neural regulation of gastrointestinal motility.NEW & NOTEWORTHY This is the first study investigating the role of nitric oxide (NO) and intramuscular interstitial cells of Cajal (ICC-IM) in modulating neural release of purines in colon. We found that NO inhibited release of purines in human, monkey, and murine colons and that colons from KitW/KitW-v (W/Wv ) mice, which present with partial loss of ICC-IM, demonstrated augmented neural release of purines. Interactions between nitrergic and purinergic neurotransmission may affect motility in disease conditions with ICC-IM deficiencies.

Cutaneous metastasis of transitional cell carcinoma of the urinary bladder eight years after the primary: a case report.

INTRODUCTION: Cutaneous metastasis of bladder carcinoma is extremely rare with a limited number of published cases. An awareness of this rare clinical entity and high index of suspicion is needed for diagnosis, as it can occur months or rarely as in this case, even years, after the primary cancer. CASE PRESENTATION: An 81-year-old Caucasian man presented with a one-year history of increasing left leg swelling and a two-month history of a macular-nodular rash on the anterior thigh, on a background of a high-grade (WHO Grade 2 of 3) papillary and invasive transitional cell carcinoma of the bladder in 2006. Following investigations, he was diagnosed as having probable locoregional recurrence of previously resected urothelial cancer of the bladder with extensive retrograde lymphatic permeation into the left thigh with cutaneous eruptions of malignancy. He completed a planned course of palliative radiation therapy to the left thigh lesions (30 Gy divided over 10 fractions) as well as the left pelvic node (a total dose of 18 Gy divided over six fractions). The disease ran an aggressive course and our patient died six months after the diagnosis of cutaneous metastases. CONCLUSIONS: Metastatic disease should always be considered in the differential diagnosis in patients with a previous history of bladder cancer who present with cutaneous nodules, even many years after the initial diagnosis at the primary site.

Retrospective study of seizure-related injuries in older people: a 10-year observation.

OBJECTIVE: The goal of this study was to assess the characteristics and risk factors for injuries caused by seizures in older persons. METHODS: All patients aged 65 years or older having injuries secondary to seizures between July 1, 1999, and June 30, 2009, were identified through the diagnostic coding system. RESULTS: Over the assessment period, a total of 615 patient episodes of seizures were recorded. Nineteen seizure-related injury events occurred in 18 persons with a total of 31 injuries. In the control group, 34 non-seizure-related injuries that were not seizure related occurred in 27 patients (28 patient episodes) (P = 0.21). The majority of injuries in both groups resulted from falls. Fifty-three percent of seizures occurred indoors, and the majority of seizures were generalized tonic-clonic seizures. Of the seizure-related injury events, 8 (42%) were single injuries, and 11 (61%) were multiple injuries. The predominant injuries were soft tissue injuries (55%), fractures (35%), and head injuries (10%). The groups did not differ significantly with respect to fractures (P = 0.06) or soft tissue injuries with lacerations (P = 0.41), or injury severity (P = 0.16), or treatment of osteoporosis (P = 0.56). CONCLUSION: These findings suggest that falls rather than seizures per se are the dominant influence in the pathogenesis of fractures in older patients with epilepsy.

Management dilemma; a woman with cystic fibrosis and severe lung disease presenting with colonic carcinoma: a case report.

INTRODUCTION: There are increasing reports of bowel cancer in cystic fibrosis, suggesting a possible causal link. Individuals with cystic fibrosis who have advanced lung disease present a high operative risk, limiting curative treatment options in early bowel malignancy. CASE PRESENTATION: We describe a 41-year-old Caucasian woman with cystic fibrosis and severe lung disease who had been considered for lung transplantation, who presented with rectal bleeding and was found to have a Stage I adenocarcinoma of the sigmoid colon. After considerable discussion as to the operative risks, she underwent a laparoscopic resection and remains relatively well 1 year postoperatively with no recurrence. CONCLUSION: We discuss the complexity of the management decisions for cystic fibrosis patients with severe lung disease and early stage colonic malignancy, particularly in the context of potential need for lung transplantation. The case demonstrates that cystic fibrosis patients with very severe lung function impairment may undergo laparoscopic abdominal surgical interventions without compromising postoperative airway clearance.

Health anxiety, anxiety sensitivity, and attentional biases for pictorial and linguistic health-threat cues.

The study investigated attentional biases for pictorial and linguistic health-threat stimuli in high and low health anxious individuals, who were selected from the upper and lower quartile ranges of a normal sample using a screening measure of health anxiety. Attentional bias was assessed using a visual probe task which presented health-threat and neutral pictures and words at two exposure durations, 500 ms and 1250 ms. The prediction that the high health anxious group would show a greater attentional bias for health-threat cues than the low health anxious group was not supported despite the groups being well-differentiated on a general measure of health anxiety, the Illness Attitudes Scale (IAS). Instead, the results indicated that individuals with high levels of anxiety sensitivity showed a significantly greater initial attentional bias for threat pictures compared with those with low anxiety sensitivity, as assessed by the Anxiety Sensitivity Index (ASI).