Aegera Therapeutics (Canada)

Abstract Background: Elevated TRIM37 protein levels, often driven by genomic copy number (CN) gains in cancer cells, lead to loss of the pericentriolar material during mitosis and increased dependence on PLK4-mediated centriole assembly. This synthetic lethal hypothesis is being tested in a phase I clinical trial evaluating the activity of the PLK4 inhibitor, RP-1664, in advanced solid tumors with TRIM37 CN gains or overexpression (LIONS, NCT06232408). In support of this clinical study, we characterized TRIM37 CN and RNA expression across a large genomic database and by imaging-based DNA and RNA in-situ hybridization. Methods: Whole exomes and transcriptomes from >12,000 adult solid tumors were analyzed for TRIM37 CN gain (CN > ploidy) or amplification (amp, CN ≥ ploidy + 2), respectively, or TRIM37 RNA expression (BostonGeneTM). Locus specific probes for fluorescence in-situ hybridization (FISH) were developed for TRIM37 and centromere 17 (CEP17). TRIM37 RNA-ISH was developed using the RNAscopeTM system (ACD BioTM). Results: Across adult solid tumors, TRIM37 amps, were most frequent in breast (9%), lung adenocarcinoma (5%), and melanoma (5%). Using the lower cutoff inclusive of TRIM37 gains, CN events were most often observed in urinary tract cancer (33%), lung adenocarcinoma (32.2%), breast cancer (26%), and squamous cell lung carcinoma (24.2%). TRIM37 CN levels were correlated with TRIM37 RNA expression in all indications examined. TRIM37 CN level was inversely correlated with segment length (coef -1.76, p-value = 6.1e-20). ERBB2 (HER2) is located approximately 19 megabases (mb) from TRIM37 and was often co-amplified, with 73% of ERBB2 gain/amps also having TRIM37 gain/amps. TRIM37 focal amps (<5.6 mb) were enriched in samples with co-occurring ERBB2 focal amps, even though these events were on different segments. HER2-enriched breast cancer had the highest percentage of focal TRIM37 amps (56%) and high-level amps (≥ 5 copies; 60%). To identify TRIM37 high patients clinically, we developed RNA and DNA ISH assays. TRIM37 DNA FISH ratio and ploidy corrected CN by NGS were strongly correlated (N=16, Pearson correlation = 0.89, p-value = 3.4x10-6). DNA FISH ratio was positively associated with RNA-ISH scores (Jonckheere-Terpstra test, P = 0.03), and of 16 TRIM37 FISH positive DNA-FISH samples, 14 were scored 2-3+ (≥4 copies/cell), while 2, scored 1+ (1-3 copies per cell) by RNA-ISH. Conclusions: A considerable population of adult solid tumors are TRIM37-high. Creation of fit-for-screening tools employing in-situ hybridization techniques may provide enhanced sensitivity for identification of patients with TRIM37-high tumors. Citation Format: Isabel Soria-Bretones,Joseph D. Schonhoft,Esha Jain,Nikita Kotlov,Parham Nejad,Daria F. Goncharova,Oleg A. Baranov,Jessica K. Scher,Elizabeth A. Sheehan,Vladimir A. Kushnarev,Konstantin Chernyshov,Gary Marshall,Ian M. Silverman,Victoria Rimkunas. Pan-cancer analysis of TRIM37 copy-number and development of fit-for-screening in situ hybridization tools [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 717.

BackgroundSuperior treatment outcomes were observed with the β-lactam/β-lactamase inhibitor combination of cefepime-enmetazobactam (FPE) compared to piperacillin-tazobactam (PTZ) in the primary efficacy population (m-MITT) of the ALLIUM phase 3 study of adult patients with cUTI/AP. We present here the outcomes in the microbiologically evaluable (ME) and ME+Resistant (ME+R) populations.Methods1034 cUTI/AP patients randomized 1:1 in a double-blind, multicenter trial received either 2 g cefepime/0.5 g enmetazobactam or 4 g piperacillin/0.5 g tazobactam q8h by 2h infusion for 7 to 14 days. Patients in m-MITT had a Gram-negative urinary baseline pathogen (BP) at >105 CFU/ml with FPE MIC ≤8 µg/ml and PTZ MIC ≤64 µg/ml. ME included patients in m-MITT who received ≥15 consecutive doses of study drug or were classified as clinical failures after receiving ≥9 doses; had a clinical assessment at test-of-cure (TOC) unless clinical failure occurred earlier; did not receive concomitant antibiotics with a non-study agent; and did not have any other protocol violation. ME+R included patients in ME along with those who had BP resistant to either FPE (MIC >8 µg/ml) or PTZ (MIC >64 µg/ml), or a missing MIC value. Overall success was the composite of clinical cure and microbiological eradication (< 103 CFU/ml in urine). Two-sided 95% confidence interval (CI) were computed using the stratified Newcombe method.ResultsIn the ME population, superiority in overall success of FPE (87.0%; 268/308) compared to PTZ (65.4%; 195/298) was demonstrated as the lower bound of the CI (16.6%) of the treatment difference (TD; 23.3%) was greater than 0 (Table). Higher rates of microbiological eradication with FPE contributed to the superior treatment outcomes. In the ME+R population in which BP susceptibility was not an exclusion criterion, favorable outcomes with FPE in overall success (TD 21.6%; 95% CI [15.3, 27.8]) and microbiological eradication (TD 21.0%; 95% CI [14.8, 27.0]) were also observed.ConclusionThe confirmation of superior treatment outcomes with FPE in the ME and ME+R populations supports the robustness of the corresponding superiority observed in adult cUTI/AP patients in m-MITT.DisclosuresAdam Belley, PhD, Allecra Therapeutics SAS (Consultant) Philip Barth, MD, Allecra Therapeutics SAS (Consultant) Omar Lahlou, PhD, Allecra Therapeutics SAS (Employee) Patrick Velicitat, MD, Allecra Therapeutics SAS (Employee)

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2–4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD–exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

G-protein-coupled receptors (GPCRs) have proven to be highly successful therapeutic targets, representing more than 40% of the top 200 worldwide best-selling drugs, and are projected to represent 120 billion US dollars of revenues per year by 2017. Interestingly, these marketed compounds modulate the activity of a restricted set of 100 GPCRs out of the 350 that are considered to be potential drug targets, indicating that in spite of the powerful drug discovery arsenal that is now available, it remains hard to “crack” new GPCRs. This is probably due to the inherent complexity of GPCR signaling, that arises from the multiplicity of intracellular pathways that can be elicited by a single receptor, according to the downstream effectors it is interacting with. To overcome these obstacles, an interesting strategy has emerged targeting allosteric modulator (AM) sites. This chapter reviews current technologies used for AM high-throughput screening (HTS) and signaling characterization, as well as the utility of combining novel approaches to efficiently discover and characterize their ability to activate or inhibit specific signaling pathways.

We have developed a strategy for synthesizing passively permeable peptidomimetic macrocycles. The cyclization chemistry centers on using aziridine aldehydes in a multicomponent reaction with peptides and isocyanides. The linker region in the resulting product contains an exocyclic amide positioned α to the peptide backbone, an arrangement that is not found among natural amino acids. This amide provides structural rigidity within the cyclic peptidomimetic and promotes the creation of a stabilizing intramolecular hydrogen bonding network. This exocyclic control element also contributes to the increased membrane permeability exhibited by multicomponent-derived macrocycles with respect to their homodetic counterparts. The exocyclic control element is employed along with a strategic placement of N-methyl and d-amino acids to produce passively permeable peptides, which contain multiple polar residues. This strategy should be applicable in the pursuit of synthesizing therapeutically relevant macrocycles.

Our previously reported structures of calpain bound to its endogenous inhibitor calpastatin have motivated the use of aziridine aldehyde-mediated peptide macrocyclization toward the design of cyclic peptides and peptidomimetics as calpain inhibitors. Inspired by nature's hint that a β-turn loop within calpastatin forms a broad interaction around calpain's active site cysteine, we have constructed and tested a library of 45 peptidic compounds based on this loop sequence. Four molecules have shown reproducibly low micromolar inhibition of calpain-2. Further systematic sequence changes led to the development of probes that displayed increased potency and specificity of inhibition against calpain over other cysteine proteases. Calculated Ki values were in the low micromolar range, rivaling other peptidomimetic calpain inhibitors and presenting an improved selectivity profile against other therapeutically relevant proteases. Competitive and mixed inhibition against calpain-2 was observed, and an allosteric inhibition site on the enzyme was identified for a noncompetitive inhibitor.

The factors determining diastereoselectivity observed in the multicomponent conversion of amino acids, aziridine aldehyde dimers, and isocyanides into chiral piperazinones have been investigated. Amino acid-dependent selectivity for either trans- or cis-substituted piperazinone products has been achieved. An experimentally determined diastereoselectivity model for the three-component reaction driven by aziridine aldehyde dimers has predictive value for different substrate classes. Moreover, this model is useful in reconciling the previously reported observations in multicomponent reactions between isocyanides, α-amino acids, and monofunctional aldehydes.

The primary sigma factor of Staphylococcus aureus, sigma(SA), regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with sigma(SA) both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of sigma(SA) that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-sigma factor may occlude the -35 element recognition domain of sigma(SA). As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of sigma(SA) to DNA and sigma(SA)-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.