Triple-negative breast cancer (TNBC) is a heterogeneous disease. We previously showed that homologous recombination deficiency (HRD) and the DNA damage immune response (DDIR) signature are prognostic in TNBC. We hypothesized that these biomarkers reflect related but not completely interdependent biological processes, that their combined use would be prognostic, and that simultaneous assessment of the immunologic microenvironment and susceptibility to DNA damaging therapies might be able to identify subgroups with distinct therapeutic vulnerabilities. We analyzed the dual DDIR/HRD classification in 341 patients with TNBC treated with adjuvant anthracycline-based chemotherapy on the SWOG S9313 trial and corroborated our findings in The Cancer Genome Atlas breast cancer data set. DDIR/HRD classification is highly prognostic in TNBC and identifies biologically and immunologically distinct subgroups. Immune-enriched DDIR+/HRD+ TNBCs have the most favorable prognosis, and DDIR+/HRD- and DDIR-/HRD+ TNBCs have favorable intermediate prognosis, despite the latter being immune-depleted. DDIR-/HRD- TNBCs have the worst prognosis and represent an internally heterogeneous group of immune-depleted chemoresistant tumors. Our findings propose DDIR/HRD classification as a potentially clinically relevant approach to categorize tumors on the basis of therapeutic vulnerabilities.

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

AbstractCyclobutenes are highly strained ring systems of considerable synthetic interest that can be accessed via cycloaddition reactions between alkenes and alkynes. However, their traditional preparation relies on photochemical [2+2] cycloadditions that exploit low-wavelength UV radiation emitted from inefficient medium-pressure Hg lamps. This paper reports on the development of a modern approach using a high-power LED set-up emitting at the boundary of UV-A and visible light in conjunction with a continuous-flow reactor. The resulting flow process renders a series of cyclobutenes from maleimides and various commercial alkynes. This provides a more energy-efficient approach that is readily scalable to access multigram quantities of cyclobutenes in high chemical yields and short residence times. The value of these products is exemplified by flow-based hydrogenations yielding highly substituted cyclobutanes which represent sought after building blocks in modern medicinal chemistry programs.

Rationale: Phosphodiesterase-4 (PDE4) inhibitors have demonstrated increased efficacy in patients with chronic obstructive pulmonary disease who had chronic bronchitis or higher blood eosinophil counts. Further characterization of patients who are most likely to benefit is warranted. Objective: To identify determinants of response to the PDE4 inhibitor tanimilast. Methods: A PDE4 gene expression signature in blood was developed by unsupervised clustering of the ECLIPSE study dataset (ClinicalTrials.gov ID: NCT00292552; Gene Expression Omnibus Series ID: GSE76705). The signature was further evaluated using blood and sputum transcriptome data from the BIOMARKER study (NCT03004417; GSE133513), enabling validation of the association between PDE4 signaling and target biomarkers. Predictivity of the associated biomarkers against clinical response was then tested in the phase-2b PIONEER tanimilast study (NCT02986321). Measurements and Main Results: The PDE4 gene expression signature developed in the ECLIPSE dataset classified subgroups of patients associated with different PDE4 signaling in the BIOMARKER cohort with an area under the receiver operator curve of 98%. In the BIOMARKER study, serum IL-8 was the only variable that was consistently associated with PDE4 signaling, with lower levels associated with higher PDE4 activity. In the PIONEER study, the exacerbation rate reduction mediated by tanimilast treatment increased up to twofold in patients with lower IL-8 levels; 36% versus 18%, reaching statistical significance at ⩽20 pg/ml (P = 0.035). The combination with blood eosinophils ⩾150 μl-1 or chronic bronchitis provided further additive exacerbation rate reduction: 45% (P = 0.013) and 47% (P = 0.027), respectively. Conclusions: Using selected heterogeneous datasets, this analysis identifies IL-8 as an independent predictor of PDE4 inhibition, as tanimilast had a greater effect on exacerbation prevention in patients with chronic obstructive pulmonary disease who had lower baseline serum IL-8 levels. Testing of this biomarker in other datasets is warranted. Clinical trial registered with www.clinicaltrials.gov (NCT00292552 [Gene Expression Omnibus Series ID: GSE76705], NCT03004417 [GSE133513], and NCT02986321).

A strategy has been developed for the carbon-14 radiosynthesis of [14 C]-SHP-141, a 4-(7-hydroxycarbamoyl-heptanoyloxy)-benzoic acid methyl ester derivative containing a terminal hydroxamic acid. The synthesis involved four radiochemical transformations. The key step in the radiosynthesis was the conversion of the 7-[14 C]-cyano-heptanoic acid benzyloxyamide [14 C]-4 directly into the carboxylic acid derivative, 7-benzyloxycarbamoyl-[14 C]-heptanoic acid [14 C]-8 using nitrilase-113 biocatalyst. The final step involved deprotection of the benzyloxy group using catalytic hydrogenation to facilitate the release of the hydroxamic acid without cleaving the phenoxy ester. [14 C]-SHP-141 was isolated with a radiochemical purity of 90% and a specific activity of 190 μCi/mg from four radiochemical steps starting from potassium [14 C]-cyanide in a radiochemical yield of 45%.

New technologies are required to combat the challenges faced with manufacturing commercial quantities of oligonucleotide drug substances which are required for treating large patient populations. Herein we report a convergent biocatalytic synthesis strategy for an Alnylam model siRNA. The siRNA chemical structure includes several of the unnatural modifications and conjugations typical of siRNA drug substances. Using Almac's 3-2-3-2 hybrid RNA ligase enzyme strategy that sequentially ligates short oligonucleotide fragments (blockmers), the target siRNA was produced to high purity at 1 mM concentration. Additional strategies were investigated including the use of polynucleotide kinase phosphorylation and the use of crude blockmer starting materials without chromatographic purification. These findings highlight a path toward a convergent synthesis of siRNAs for large-scale manufacture marrying both enzymatic liquid and classical solid-phase synthesis.

Abstract The placental alkaline phosphatases, ALPP and ALPPL2, are glycosylphosphatidylinositol (GPI)-anchored cell-surface proteins that are expressed in the placenta during fetal development but have very little expression on normal adult tissue. These proteins are over-expressed in a number of different solid tumor indications including ovarian, endometrial, gastric, pancreatic and non-small cell lung cancers. As a result of the highly restricted normal tissue expression and over-expression in cancer, ALPP and ALPPL2 are attractive targets for antibody and protein-drug conjugate (ADC and PDC) approaches. The recent initiation of a Phase I clinical trial utilizing a full-length antibody ADC has fueled further interest in these oncology targets. Small protein domain binders, which have the capacity to penetrate deeper into solid tumors and can be engineered into multiple therapeutic formats in a modular fashion, offer a number of potential benefits over full-length antibodies as the targeting vehicle in PDC therapeutics. We report the identification and characterization of a series of high affinity ALPP/ALPPL2 specific single domain VHH binders which, importantly, show no binding to the closely related ALPI or ALPL isoforms (which have high normal tissue expression). Through application of the Almac Discovery PDC technology platform, these VHH domains have been reformatted into a suite of homogenous site-specifically labelled drug conjugates, with defined drug to antibody ratios, in high yields. These conjugates, which are based on mono- and bi-paratopic Fc fusion formats, employ both clinically established and novel linker-toxin combinations. In pre-clinical studies, the lead bi-paratopic PDCs were well tolerated in vivo and showed excellent anti-tumor efficacy in ALPP/ALPPL2 positive cell-line derived xenograft models of gastric and pancreatic carcinoma, with sustained regressions still observed 10 weeks after administration of the final dose of agent. These agents have physicochemical and pharmaceutical properties suitable for further development and we anticipate that the excellent pre-clinical efficacy profile of lead PDCs will translate to a highly differentiated product for the treatment of a variety of solid tumor indications. Citation Format: Graham Cotton, Paul Trumper, Estelle McLean, Mark Wappett, Stacey Bell, Greg Papadakos, Jennifer Thom, Chiara Saladino, Aaron Cranston, Georgiana Parau, Stephanie Gatdula, Stephanie Burton, Aidan McCann, Tim Harrison. Development of novel protein drug conjugates (PDCs) for the selective targeting of ALPP/ALPPL2 expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB011.

Abstract Cancer cachexia is a metabolic wasting syndrome characterized by weight loss, anorexia and anemia as a result of tumor burden, and affects up to 80% of advanced cancer patients #1. Cachexia is particularly prevalent in pancreatic, lung, colorectal and gastro-intestinal cancers and can lead to reduced tolerance and responsiveness to chemotherapy, increased treatment-related toxicity and morbidity, and poor overall quality of life. There are currently no approved therapies for cancer cachexia. The development and maintenance of muscle tissue is dependent on the balance between protein synthesis and protein degradation, controlled through various anabolic and catabolic signaling pathways. Dysregulation of these pathways can result in muscle atrophy, which arises in many chronic illnesses. The ubiquitin proteasome system (UPS) has a central role in regulating skeletal muscle physiology. Previous work utilizing USP19 knock out mouse models has demonstrated that USP19 plays an important role in muscle wasting and can protect against denervation-induced muscle atrophy #2. We have previously demonstrated that inhibition of USP19 enzymatic activity spares the muscle wasting observed in limb-casted and denervated mouse models of muscle wasting. Here, we report the discovery of a novel, highly potent and selective inhibitor of USP19 (ADC-846) and demonstrate its utility in a cancer-induced muscle atrophy model in vivo. Pharmacological inhibition of USP19 by ADC-846 increased lean muscle and fat mass following oral dosing in a Lewis Lung Carcinoma-induced cachexia model and reduced the cachexic index by >60% compared to controls. This data, in combination with our previous work detailing the effect of USP19 inhibition on muscle force and function, provides a much-needed novel pharmacological strategy for therapeutic intervention in muscle wasting conditions. Citation Format: Natalie Page, Vignesh Karthikaisamy, Darren O'Hara, Aaron N. Cranston, Colin R. O'Dowd, Xavier Jacq, Richard Wilkinson, Stephanie Burton, Hayley Gibson, Joana Costa, Daniel Longley, Matthew Helm, Chris McGivern, Steven Shepherd, Christina Bell, Peter Hewitt, Mary McFarland, Hugues Miel, Steven Whitehead, Lauren Proctor, Shane J. Rountree, Mark Wappett, Mauricio Berriel Diaz, Stephan Herzig, Timothy Harrison. A novel first-in-class USP19 inhibitor for the treatment of cancer-induced muscle atrophy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB022.

Methionine sulfoxide reductase A (MsrA) enzymes have recently found applications as nonoxidative biocatalysts in the enantioselective kinetic resolution of racemic sulfoxides. This work describes the identification of selective and robust MsrA biocatalysts able to catalyze the enantioselective reduction of a variety of aromatic and aliphatic chiral sulfoxides at 8-64 mM concentration with high yields and excellent ees (up to 99%). Moreover, with the aim to expand the substrate scope of MsrA biocatalysts, a library of mutant enzymes has been designed via rational mutagenesis utilizing in silico docking, molecular dynamics, and structural nuclear magnetic resonance (NMR) studies. The mutant enzyme MsrA33 was found to catalyze the kinetic resolution of bulky sulfoxide substrates bearing non-methyl substituents on the sulfur atom with ees up to 99%, overcoming a significant limitation of the currently available MsrA biocatalysts.