PT-112 Induces Mitochondrial Stress and Immunogenic Cell Death, Targeting Tumor Cells with Mitochondrial Deficiencies.

PT-112 is a novel pyrophosphate-platinum conjugate, with clinical activity reported in advanced pretreated solid tumors. While PT-112 has been shown to induce robust immunogenic cell death (ICD) in vivo but only minimally bind DNA, the molecular mechanism underlying PT-112 target disruption in cancer cells is still under elucidation. The murine L929 in vitro system was used to test whether differential metabolic status alters PT-112's effects, including cell cytotoxicity. The results showed that tumor cells presenting mutations in mitochondrial DNA (mtDNA) (L929dt and L929dt cybrid cells) and reliant on glycolysis for survival were more sensitive to cell death induced by PT-112 compared to the parental and cybrid cells with an intact oxidative phosphorylation (OXPHOS) pathway (L929 and dtL929 cybrid cells). The type of cell death induced by PT-112 did not follow the classical apoptotic pathway: the general caspase inhibitor Z-VAD-fmk did not inhibit PT-112-induced cell death, alone or in combination with the necroptosis inhibitor necrostatin-1. Interestingly, PT-112 initiated autophagy in all cell lines, though this process was not complete. Autophagy is known to be associated with an integrated stress response in cancer cells and with subsequent ICD. PT-112 also induced a massive accumulation of mitochondrial reactive oxygen species, as well as changes in mitochondrial polarization-only in the sensitive cells harboring mitochondrial dysfunction-along with calreticulin cell-surface exposure consistent with ICD. PT-112 substantially reduced the amount of mitochondrial CoQ10 in L929 cells, while the basal CoQ10 levels were below our detection limits in L929dt cells, suggesting a potential relationship between a low basal level of CoQ10 and PT-112 sensitivity. Finally, the expression of HIF-1α was much higher in cells sensitive to PT-112 compared to cells with an intact OXPHOS pathway, suggesting potential clinical applications.

Comparative Effectiveness of Nonoperative Versus Operative Treatment of Completely Displaced Clavicle Shaft Fractures Among Children.

The goal of this study was to compare outcomes among children treated nonoperatively vs operatively for completely displaced clavicle fractures. This was a retrospective cohort study of nonoperative vs operative treatment of completely displaced clavicle fractures sustained between 2006 and 2015 among pediatric patients. Data were collected on patient demographics, fracture characteristics, time to return to full activities, treatment complications, and patient-reported outcome measures. Fifty-five patients were identified in the nonoperative group, with a mean age of 11.6 years (range, 8-14 years). The operative group contained 55 patients, with a mean age of 14.3 years (range, 9-17 years). All fractures healed, with a mean time to return to full activities of 90.4 days in the nonoperative group and 89.7 days in the operative group (P=.941). Twelve (22%) nonoperative patients sustained a refracture of their clavicle compared with 4 patients in the operative group (P=.031). Fifteen patients (27%) in the operative group required a second surgery for removal of surgical implants. On the shortened form of the Disabilities of the Arm, Shoulder and Hand (QuickDASH) survey, 17 of the 22 nonoperative patients reported a score of zero (indicating no disability) (range, 0-7) vs 22 of 25 in the operative group (range, 0-9) (P=.329). Patients treated nonoperatively had a 22% rate of refracture, whereas patients treated operatively had a 27% rate of undergoing a second surgery for removal of surgical implants. These data can aid in the shared decision-making process with patients and families when deciding on treatment for displaced pediatric clavicle fractures. [Orthopedics. 2022;45(6):373-377.].

Phase I study of PT-112, a novel pyrophosphate-platinum immunogenic cell death inducer, in advanced solid tumours.

Background: PT-112, the first pyrophosphate-platinum conjugate, causes immunogenic cell death in experimental models, leading to recruitment of tumour-infiltrating lymphocytes. PT-112 also associates with bone (osteotropism), likely driven by its pyrophosphate moiety. This is the first-in-human study of PT-112 monotherapy, exploring its safety and efficacy in a patient population where standard of care therapies were exhausted and novel treatment options are needed. Methods: Patients with progressing, advanced solid tumours received PT-112 intravenously (1 h) on days 1, 8, 15 of a 28-day cycle in an open-label, multi-centre 3 + 3 dose-escalation trial, conducted at four US research sites. The primary objective was to assess safety and pharmacokinetics, and to identify a recommended phase 2 dose (RP2D). Eligibility criteria included: age ≥18 years, Eastern Collaborative Oncology Group (ECOG) Performance Status of 0-1, and disease evaluable by Response Evaluation Criteria in Solid Tumours (RECIST) v1·1 or by informative tumour markers. Patients receiving ≥1 dose of PT-112 were included in the safety and pharmacokinetic analyses, with the exploratory efficacy analysis including patients receiving ≥1 dose at 125 mg/m2. This study is registered at ClinicalTrials.gov, number NCT02266745, with the dose-escalation portion of the study closed. Findings: Between July 7th, 2014 and September 18th, 2018, 66 heavily pre-treated patients (median 4 prior lines, IQR 2-6) were enrolled and treated across 11 doses (12-420 mg/m2). Treatment-related adverse events included fatigue (23 patients, 35%), nausea (16 patients, 24%), and peripheral neuropathy (14 patients, 21%). Grade 3 events were experienced by 18 patients (27%), with no grade 4-5 events observed. The recommended phase 2 dose was determined to be 360 mg/m2. Nine (17%) of the 54 efficacy evaluable patients achieved progression-free survival ≥6 months. Durable partial responses were induced in non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and thymoma. Radiographic and serum marker reductions were observed among ten patients with metastatic castration resistant prostate cancer, four of whom survived two years or longer. Interpretation: PT-112 is safe and well-tolerated in a heavily pre-treated population. Prolonged responses were noted against thymoma and lung cancer, along with radiographic and serum marker improvement in prostate cancer. Given the heterogeneous patient population, subsequent studies will be needed to characterize the risk/benefit ratio in more homogenous settings. Further development of PT-112 is ongoing, as single-agent and in combination with immune checkpoint inhibition. Funding: Funding was provided by Promontory Therapeutics Inc.

PT-112 induces immunogenic cell death and synergizes with immune checkpoint blockers in mouse tumor models.

PT-112 is a novel platinum-pyrophosphate conjugate under clinical development for cancer therapy. PT-112 mediates cytostatic and cytotoxic effects against a variety of human and mouse cancer cell lines in vitro. The cytotoxic response to PT-112 is associated with the emission of danger signals underpinning the initiation of anticancer immunity, including calreticulin exposure on the surface of dying cells, as well as ATP and HMGB1 secretion. Consistently, mouse cancer cells succumbing to PT-112 in vitro can be used to provide syngeneic, immunocompetent mice with immunological protection against a subsequent challenge with living tumor cells of the same type. Moreover, PT-112 administration synergizes with PD-1 or PD-L1 blockade in the control of mouse cancers in immunologically competent settings, as it simultaneously recruits immune effector cells and depletes immunosuppressive cells in the tumor microenvironment. Finally, PT-112 employed intratumorally in the context of immune checkpoint inhibition initiates a robust immune response that has systemic outreach and limits the growth of untreated, distant lesions. Thus, PT-112 induces the immunogenic demise of cancer cells, and hence stands out as a promising combinatorial partner of immune checkpoint blockers, especially for the treatment of otherwise immunologically cold tumors.

Detection of 224 candidate structured RNAs by comparative analysis of specific subsets of intergenic regions.

The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

Quantitative Trait Loci (QTL)-Guided Metabolic Engineering of a Complex Trait.

Engineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness of Saccharomyces cerevisiae during fermentation of a Miscanthus x giganteus plant hydrolysate. We first used high-resolution Quantitative Trait Loci (QTL) mapping and systematic bulk Reciprocal Hemizygosity Analysis (bRHA) to discover 17 loci that differentiate hydrolysate tolerance between an industrially related (JAY291) and a laboratory (S288C) strain. We then used this data to identify a subset of favorable allelic loci that were most amenable for strain engineering. Guided by this "genetic blueprint", and using a dual-guide Cas9-based method to efficiently perform multikilobase locus replacements, we engineered an S288C-derived strain with superior hydrolysate tolerance than JAY291. Our methods should be generalizable to engineering any complex trait in S. cerevisiae, as well as other organisms.

Fear of Falling Correlates with Subtle Neuromuscular Balance and Strength Deficits of Fragility Fracture Patients.

Fragility fractures, or fractures occurring from a low-trauma event, are extremely prevalent among the elderly population worldwide and associated with significant mortality and morbidity. This study evaluated the relationship between FES-I Fear of Falling Survey results, self-reported activity restrictions via the SF-36 survey, and scores recorded by portable, inexpensive clinical assessment tools (CATs) during dynamic functional tasks. Low scores during these tasks may indicate functional deficits that put patients at risk for falls and subsequent fragility fractures. Forty-one subjects (20 fragility fracture patients, 21 controls without history of fragility fractures) over the age of 50 were recruited from three outpatient orthopaedic clinics. All subjects were administered a FES-I Fear of Falling Survey, a portion of an SF-36 survey, and tested using three different portable CATs: the Wii Balance Board, iPod Level Belt and Saehan Squeeze Hand Grip Dynamometer. There were several measured variables that showed a moderate correlation with Fear of Falling scores. Of note, correlations between FES-I scores and maximum hand grip strength for both the dominant hand (R= -0.302, p=0.069) and non-dominant hand (R= -0.309, p=0.059), as well as maximum anterior-posterior sway measured by the iPod Level Belt (R=0.320, p=0.056) were found to be marginally significant. In addition, the correlation between FES-I and average anterior-posterior sway was found to be significant (R=0.416, p=0.012). The Nintendo Wii and iPod Level Belt are relatively inexpensive, portable tools that can assess patients for subtle deficits during dynamic functional tasks. The results indicate that these tools can provide a more objective measure of a patient's limitations during daily activities such as walking by assigning them a numerical value and correlating this value to physical deficits that impact balance and coordination. In the future, CATs may also have a role in predicting outcomes and in individualizing care, therapy, and at-home preventive measures.

Clinical assessment tools identify functional deficits in fragility fracture patients.

PURPOSE: To identify inexpensive, noninvasive, portable, clinical assessment tools that can be used to assess functional performance measures that may put older patients at risk for falls such as balance, handgrip strength, and lumbopelvic control. PATIENTS AND METHODS: Twenty fragility fracture patients and 21 healthy control subjects were evaluated using clinical assessment tools (Nintendo Wii Balance Board [WBB], a handheld dynamometer, and an application for the Apple iPod Touch, the Level Belt) that measure functional performance during activity of daily living tasks. The main outcome measurements were balance (WBB), handgrip strength (handheld dynamometer), and lumbopelvic control (iPod Touch Level Belt), which were compared between fragility fracture patients and healthy controls. RESULTS: Fragility fracture patients had lower scores on the vertical component of the WBB Torso Twist task (P=0.042) and greater medial-lateral lumbopelvic sway during a 40 m walk (P=0.026) when compared to healthy controls. Unexpectedly, the fracture patients had significantly higher scores on the left leg (P=0.020) and total components (P=0.010) of the WBB Single Leg Stand task as well as less faults during the left Single Leg Stand task (P=0.003). CONCLUSION: The clinical assessment tools utilized in this study are relatively inexpensive and portable tools of performance measures capable of detecting differences in postural sway between fragility fracture patients and controls.

Training Advanced Practice Providers to Collect Functional Outcomes After Fragility Fractures.

OBJECTIVE: The objective of this study was to determine whether advanced practice providers could learn to collect objective functional assessment data accurately and efficiently with commercially available devices that measure kinematics and kinetics (Nintendo Wii Balance Board [WBB] and Level Belt [LB]) to aid in the assessment of fall risk and outcomes after fragility fractures. METHODS: Nine advanced practice providers participated in a 1-hour clinical assessment tools (CATs) training session on equipment use, providing standardized instructions, and practice of the testing procedures. Afterward, they participated in a skills demonstration evaluation and completed a postsession survey. RESULTS: Participants successfully achieved a mean of 18.22 (standard deviation 1.56) of 20 performance measures. Of the incomplete or omitted tasks, the majority (10 of 16) occurred within the first of 3 CATs activities. Postsession survey results revealed that 9 of 9 participants reported that the 1 hour provided for training on the CATs was sufficient. All participants reported that after the training, they felt confident they could reliably carry out the tasks to test patients on both the WBB and the LB. The majority of participants reported that they believed that the WBB (7 of 9) and LB (8 out of 9) would be good assets to clinics in assessing patient functionality after fragility fractures. CONCLUSION: These results indicate that advanced practice providers can confidently learn and effectively test patients with the WBB and LB within 1 hour of training. In the future, adoption of CATs in the clinical setting may allow for objective, easy-to-use, portable, noninvasive, and relatively inexpensive measures to assess functional outcomes in patients with fragility fracture.

Small molecule fluoride toxicity agonists.

Fluoride is a ubiquitous anion that inhibits a wide variety of metabolic processes. Here, we report the identification of a series of compounds that enhance fluoride toxicity in Escherichia coli and Streptococcus mutans. These molecules were isolated by using a high-throughput screen (HTS) for compounds that increase intracellular fluoride levels as determined via a fluoride riboswitch reporter fusion construct. A series of derivatives were synthesized to examine structure-activity relationships, leading to the identification of compounds with improved activity. Thus, we demonstrate that small molecule fluoride toxicity agonists can be identified by HTS from existing chemical libraries by exploiting a natural fluoride riboswitch. In addition, our findings suggest that some molecules might be further optimized to function as binary antibacterial agents when combined with fluoride.

A widespread self-cleaving ribozyme class is revealed by bioinformatics.

Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2,700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.

Bacterial aptamers that selectively bind glutamine.

The continued expansion of microbial sequence data has allowed for the detection of an increasing number of conserved RNA motifs by using comparative sequence analysis. Recently, we reported the discovery of two structured non-coding RNA motifs, called glnA and Downstream-peptide, that have similarity in sequence and secondary structure. In this report, we describe data demonstrating that representatives of both RNA motifs selectively bind the amino acid L-glutamine. These glutamine aptamers are found exclusively in cyanobacteria and marine metagenomic sequences, wherein several glnA RNA representatives reside upstream of genes involved in nitrogen metabolism. These motifs have genomic distributions that are consistent with a gene regulation function, suggesting they are components of glutamine-responsive riboswitches. Thus, our findings implicate glutamine as a regulator of cyanobacterial nitrogen metabolism pathways. Furthermore, our findings expand the collection of natural aptamer classes that bind amino acids to include glycine, lysine and glutamine.

A eubacterial riboswitch class that senses the coenzyme tetrahydrofolate.

Comparative sequence analyses of bacterial genomes are revealing many structured RNA motifs that function as metabolite-binding riboswitches. We have identified an RNA motif frequently positioned in the 5' UTRs of folate transport and biosynthesis genes in Firmicute genomes. Biochemical experiments confirm that representatives of this new-found RNA class selectively bind derivatives of the vitamin folate, including di- and tetrahydrofolate coenzymes. In addition, representatives of this aptamer class occasionally reside upstream of RNA structures that are predicted to control translation initiation in response to ligand binding. These findings expand the number of coenzymes that are directly sensed by RNA and reveal possible riboswitch-controlled regulons that respond to changes in single-carbon metabolism.

Structural basis of ligand binding by a c-di-GMP riboswitch.

The second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP-binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 A resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.

A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria.

Riboswitches that sense S-adenosylmethionine (SAM) are widely distributed throughout a variety of bacterial lineages. Four classes of SAM-binding riboswitches have been reported to date, constituting the most diverse collection of riboswitch classes that sense the same compound. Three of these classes, termed SAM-I, SAM-II, and SAM-III represent unique structures that form distinct binding pockets for the ligand. SAM-IV riboswitches carry different conserved sequence and structural features compared to other SAM riboswitches, but nucleotides and substructures corresponding to the ligand binding pocket are identical to SAM-I aptamers. In this article, we describe a fifth class of SAM binding aptamer, which we have termed SAM-V. SAM-V was discovered by analyzing GC-rich intergenic regions preceding metabolic genes in the marine alpha-proteobacterium "Candidatus Pelagibacter ubique." Although the motif is nearly unrepresented in cultured bacteria whose genomes have been completely sequenced, SAM-V is prevalent in marine metagenomic sequences. The consensus sequence and structure of SAM-V show some similarities to that of the SAM-II riboswitch, and it is likely that the two aptamers form similar ligand binding pockets. In addition, we identified numerous examples of a tandem SAM-II/SAM-V aptamer architecture. In this arrangement, the SAM-II aptamer is always positioned 5' of the SAM-V aptamer and the SAM-II aptamer is followed by a predicted intrinsic transcription terminator stem. The SAM-V aptamer, however, appears to use a ribosome binding site occlusion mechanism for genetic regulation. This tandem riboswitch arrangement exhibits an architecture that can potentially control both the transcriptional and translational stages of gene expression.

Identification of candidate structured RNAs in the marine organism 'Candidatus Pelagibacter ubique'.

BACKGROUND: Metagenomic sequence data are proving to be a vast resource for the discovery of biological components. Yet analysis of this data to identify functional RNAs lags behind efforts to characterize protein diversity. The genome of 'Candidatus Pelagibacter ubique' HTCC 1062 is the closest match for approximately 20% of marine metagenomic sequence reads. It is also small, contains little non-coding DNA, and has strikingly low GC content. RESULTS: To aid the discovery of RNA motifs within the marine metagenome we exploited the genomic properties of 'Cand. P. ubique' by targeting our search to long intergenic regions (IGRs) with relatively high GC content. Analysis of known RNAs (rRNA, tRNA, riboswitches etc.) shows that structured RNAs are significantly enriched in such IGRs. To identify additional candidate structured RNAs, we examined other IGRs with similar characteristics from 'Cand. P. ubique' using comparative genomics approaches in conjunction with marine metagenomic data. Employing this strategy, we discovered four candidate structured RNAs including a new riboswitch class as well as three additional likely cis-regulatory elements that precede genes encoding ribosomal proteins S2 and S12, and the cytoplasmic protein component of the signal recognition particle. We also describe four additional potential RNA motifs with few or no examples occurring outside the metagenomic data. CONCLUSION: This work begins the process of identifying functional RNA motifs present in the metagenomic data and illustrates how existing completed genomes may be used to aid in this task.

Engineering high-speed allosteric hammerhead ribozymes.

Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg(2+). The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k (obs)) as high as 8 min(-1) in the presence of theophylline and maximal k (obs) increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.