Macrophages are critical regulators of the tumor microenvironment and often present an immuno-suppressive phenotype, supporting tumor growth and immune evasion. Promoting a robust pro-inflammatory macrophage phenotype has emerged as a therapeutic modality that supports tumor clearance, including through synergy with immune checkpoint therapies. Polyglucose nanoparticles (macrins), which possess high macrophage affinity, are useful vehicles for delivering drugs to macrophages, potentially altering their phenotype. Here, we examine the potential of functionalized macrins, synthesized by crosslinking carboxymethyl dextran with L-lysine, as effective carriers of immuno-stimulatory drugs to tumor-associated macrophages (TAMs). Azide groups incorporated during particle synthesis provided a handle for click-coupling of propargyl-modified β-cyclodextrin to macrins under mild conditions. Fluorescence-based competitive binding assays revealed the ability of β-cyclodextrin to non-covalently bind to hydrophobic immuno-stimulatory drug candidates (Keq ~ 103 M-1), enabling drug loading within nanoparticles. Furthermore, transcriptional profiles of macrophages indicated robust pro-inflammatory reprogramming (elevated Nos2 and Il12; suppressed Arg1 and Mrc1 expression levels) for a subset of these immuno-stimulatory agents (UNC2025 and R848). Loading of R848 into the modified macrins improved the drug’s effect on primary murine macrophages by three-fold in vitro. Intravital microscopy in IL-12-eYFP reporter mice (24 h post-injection) revealed a two-fold enhancement in mean YFP fluorescence intensity in macrophages targeted with R848-loaded macrins, relative to vehicle controls, validating the desired pro-inflammatory reprogramming of TAMs in vivo by cell-targeted drug delivery. Finally, in an intradermal MC38 tumor model, cyclodextrin-modified macrin NPs loaded with immunostimulatory drugs significantly reduced tumor growth. Therefore, efficient and effective repolarization of tumor-associated macrophages to an M1-like phenotype—via drug-loaded macrins—inhibits tumor growth and may be useful as an adjuvant to existing immune checkpoint therapies.

Abstract Phenomics-enabled drug discovery is a powerful approach to identify novel targets and relationships previously unappreciated in biology. We built a phenomics platform leveraging high content microscopy and generated unique phenoprints for >7,000 genes using CRISPR/Cas9 technology and a diverse chemical library of >1 million compounds. This phenomics dataset contains an unprecedented quantity of gene-gene, gene-compound, and compound-compound relationships. We applied our phenomics platform and an inference based target-agnostic drug discovery approach to discover novel genes and small molecules that mimic CDK12 inhibition, an important transcriptional regulator of DNA damage response, while avoiding inhibition of the paralog CDK13. We discovered a novel and unappreciated association between CDK12 and RBM39 as well as small molecule degraders of RBM39. We established structure activity relationships (SAR) solely leveraging our phenomics platform, to generate REC-1170204, an RBM39 degrader. Unlike inhibitors of CDK12, we show that REC-1170204 does not directly inhibit CDK12, CDK13 or other kinases. However, REC-1170204 demonstrates candidate quality properties with improved potency, selectivity, and drug-like characteristics. Finally we show that REC-1170204 shows efficacy in High Grade Serous Ovarian cancer (HGSOC) pre-clinical models and synergizes with PARP inhibition in a PARP-resistant patient-derived xenograft (PDX) model. Together, our data suggests that targeting RBM39 may provide an attractive and safer approach to targeting CDK12 in the clinic. Citation Format: Chase Neumann, Harish Shankaran, Kiran Nadella, Kelly Biette, Shane Rowley, Ethan Gardner, Shadi Swaidani, Vamshi Manda, Lu Chen, Daria Beshnova, Ashraf Saeed, Christopher Bailey, Janet Paulsen, Paul Rearden, Carl Brooks, Ashish Bhandari, Chris Gibson, Laura Schaevitz, Imran Haque, Hayley Donnella, Michael Cuccarese, Marie Evangelista. Phenomics-enabled discovery and optimization of small-molecule RBM39 degraders as an alternative to CDK12 targeting in high-grade serous ovarian cancer (HGSOC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7133.

The emergence of technological innovations has created the opportunity to envision new approaches to discover therapeutics at scale. We combined advances in high content microscopy with arrayed CRISPR genome editing techniques and machine learning (ML) to build a rigorously controlled dataset enabling exploration of biology and chemistry at scale. Phenotypes from millions of perturbations in multiple cell types were embedded in a unified representation space and leveraged to accelerate discovery and reverse translation, ultimately yielding novel biological insights, and optimizing the advancement of lead molecular series through structure-activity relationships (SAR). Here, we demonstrate the capability of our platform to discover potential cancer therapies with distinct mechanisms of action. First, we describe the identification of a novel compound series that potentiates the effects of immunotherapy in syngeneic mouse models, producing complete responses and immunological memory, while also limiting peripheral inflammation. Specific novel chemical entities (NCEs) caused robust CD45+ cell influx into the tumor microenvironment and significantly attenuated exhausted T cells and immunosuppressive macrophages, thereby enhancing anti-tumor immunity. Strikingly, the same NCEs suppressed peripheral inflammation while sustaining elevated levels of intra-tumoral proinflammatory cytokines. Second, we highlight a novel and differentiated strategy to potentiate PARP inhibitor response in homologous repair deficient (HRD) - negative or HR-proficient ovarian cancers. NCEs altered the expression of genes within the DNA damage repair (DDR) network and cell cycle checkpoints to synergize with PARP inhibition in vivo and re-sensitized a PARP-resistant patient-derived xenograft (PDX) model. Collectively, we believe future efforts on the industrialization and integration of various technological innovations across biology, chemistry, automation, data science, and engineering will ultimately modernize drug discovery and radically improve patient lives. Citation Format: Jenny Rudnick, Kiran Nadella, Chase Neumann, Shane Rowley, Ethan Gardner, Shadi Swaidani, Aimee Iberg, Lu Chen, Daria Beshnova, Aurora Blucher, Rebecca Sarto Basso, Malini Rajan, Kevin Fales, Ashraf Saeed, Christopher Bailey, Weston Judd, Chrissy Egbert, Joel Ellis, John Ansede, Pouya Hadipour, Kevin Jessing, Janet Paulsen, Paul Rearden, Vamshi Manda, Sashi Kasimsetty, Sashi Kasimsetty, Michael Hancock, Harish Shankaran, Bryan Ellis, Meenakshy Iyer, Carl Brooks, Ashish Bhandari, Chris Gibson, Irit Rappley, Laura Schaevitz, Imran Haque, Hayley Donnella, Michael Cuccarese, Marie Evangelista. A phenomics platform combining imaging and artificial intelligence for rapid validation and advancement of novel oncology targets [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 LB071.

Supplementary Data from Macrophage-Targeted Therapy Unlocks Antitumoral Cross-talk between IFNγ-Secreting Lymphocytes and IL12-Producing Dendritic Cells

Abstract Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches. We showed that tumor control was not caused by CSF1R+ cell depletion; instead, CSF1R targeting reshaped the CSF1R+ cell landscape, which unlocked cross-talk between antitumoral CSF1R− cells. These cells included IFNγ-producing natural killer and T cells, and an IL12-producing dendritic cell subset, denoted as DC3, which were all necessary for CSF1R inhibitor–mediated lung tumor control. These data indicate that CSF1R targeting can activate a cardinal cross-talk between cells that are not macrophages and that are essential to mediate the effects of T cell–targeted immunotherapies and promote antitumor immunity. See related Spotlight by Burrello and de Visser, p. 4.

Immune checkpoint inhibitors have revolutionized cancer treatment, producing a durable response consistent with immunologic memory in a subset of patients. However, the majority of patients demonstrate innate or acquired resistance that must be characterized and overcome to induce successful treatment. Advancements in human reverse translation and scaled in vivo CRISPR screening have uncovered novel molecular and genomic correlates of resistance, and promising druggable mechanisms - driven by highly complex interactions between tumor cells and the immune system. It is this core biology that must be disentangled to build the treatment paradigms of the future. Here we demonstrate a technique for massively parallel prioritization of new immuno-oncology hypotheses using industrial-scale experimentation and machine learning. Leveraging high-content imaging data from whole-genome CRISPR knockout and a library of >250,000 compounds, a deep learning model was trained to construct a batch-invariant low dimensional representation of each perturbation. Millions of perturbations in multiple cell types were embedded in a unified representation space that was leveraged to increase the rate of discovery, accelerate reverse translation, yield novel biological insights, and guide the advancement of lead molecular series through SAR. Here we highlight multiple discovery programs driven by inferred relationships between small molecules and gene knockout with translation from inference to in vivo efficacy. Specifically, we prioritize molecules with activity in STK11-deficient tumors and additional immune checkpoint sensitizers. Citation Format: Ashish Bhandari, Michael F. Cuccarese, Kevin Fales, Kiran Nadella, Rebecca Sarto Basso, Daria Beshnova, Hayley Donnella, Bahar Shamloo, Jacob Cooper, Imran Haque, Ron Alfa, Jacob Rinaldi. Identification and optimization of novel small molecule modulators of immune checkpoint resistance with a unified representation space for genomic and chemical perturbations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1888.

ABSTRACTDevelopment of accurate disease models and discovery of immune-modulating drugs is challenged by the immune system’s highly interconnected and context-dependent nature. Here we apply deep-learning-driven analysis of cellular morphology to develop a scalable “phenomics” platform and demonstrate its ability to identify dose-dependent, high-dimensional relationships among and between immunomodulators, toxins, pathogens, genetic perturbations, and small and large molecules at scale. High-throughput screening on this platform demonstrates rapid identification and triage of hits for TGF-β- and TNF-α-driven phenotypes. We deploy the platform to develop phenotypic models of active SARS-CoV-2 infection and of COVID-19-associated cytokine storm, surfacing compounds with demonstrated clinical benefit and identifying several new candidates for drug repurposing. The presented library of images, deep learning features, and compound screening data from immune profiling and COVID-19 screens serves as a deep resource for immune biology and cellular-model drug discovery with immediate impact on the COVID-19 pandemic.

Tumour-associated macrophages (TAMs) are abundant in many cancers, and often display an immune-suppressive M2-like phenotype that fosters tumour growth and promotes resistance to therapy. Yet macrophages are highly plastic and can also acquire an anti-tumourigenic M1-like phenotype. Here, we show that R848, an agonist of the toll-like receptors (TLRs) TLR7 and TLR8 identified in a morphometric-based screen, is a potent driver of the M1 phenotype in vitro and that R848-loaded β-cyclodextrin nanoparticles (CDNPs) lead to efficient drug delivery to TAMs in vivo. As a monotherapy, the administration of CDNP-R848 in multiple tumour models in mice altered the functional orientation of the tumour immune microenvironment towards an M1 phenotype, leading to controlled tumour growth and protecting the animals against tumour rechallenge. When used in combination with the immune checkpoint inhibitor anti-PD-1, we observed improved immunotherapy response rates, also in a tumour model resistant to anti-PD-1 therapy. Our findings demonstrate the ability of rationally engineered drug-nanoparticle combinations to efficiently modulate TAMs for cancer immunotherapy.

The receptor tyrosine kinase Mer (MERTK) is a promising drug target in cancer, where it can influence the metastasis-promoting signaling of both tumor cells and immune cells alike; however, no small molecule probes currently exist to selectively image Mer. In this work, we design and synthesize a selective near-infrared fluorescent molecular probe of Mer (MERi-SiR). Confocal microscopy of metastases in mice reveals predominant probe accumulation in Mer-expressing tumor-associated macrophages.

Efficient delivery of therapeutic nanoparticles (TNPs) to tumors is critical in improving efficacy, yet strategies that universally maximize tumoral targeting by TNP modification have been difficult to achieve in the clinic. Instead of focusing on TNP optimization, we show that the tumor microenvironment itself can be therapeutically primed to facilitate accumulation of multiple clinically relevant TNPs. Building on the recent finding that tumor-associated macrophages (TAM) can serve as nanoparticle drug depots, we demonstrate that local tumor irradiation substantially increases TAM relative to tumor cells and, thus, TNP delivery. High-resolution intravital imaging reveals that after radiation, TAM primarily accumulate adjacent to microvasculature, elicit dynamic bursts of extravasation, and subsequently enhance drug uptake in neighboring tumor cells. TAM depletion eliminates otherwise beneficial radiation effects on TNP accumulation and efficacy, and controls with unencapsulated drug show that radiation effects are more pronounced with TNPs. Priming with combined radiation and cyclophosphamide enhances vascular bursting and tumoral TNP concentration, in some cases leading to a sixfold increase of TNP accumulation in the tumor, reaching 6% of the injected dose per gram of tissue. Radiation therapy alters tumors for enhanced TNP delivery in a TAM-dependent fashion, and these observations have implications for the design of next-generation tumor-targeted nanomaterials and clinical trials for adjuvant strategies.

Tumor-associated macrophages limit anti–PD-1 effects by removing the antibody from CD8 + T cells.

Involvement of the immune system in tumour progression is at the forefront of cancer research. Analysis of the tumour immune microenvironment has yielded a wealth of information on tumour biology, and alterations in some immune subtypes, such as tumour-associated macrophages (TAM), can be strong prognostic indicators. Here, we use optical tissue clearing and a TAM-targeting injectable fluorescent nanoparticle (NP) to examine three-dimensional TAM composition, tumour-to-tumour heterogeneity, response to colony-stimulating factor 1 receptor (CSF-1R) blockade and nanoparticle-based drug delivery in murine pulmonary carcinoma. The method allows for rapid tumour volume assessment and spatial information on TAM infiltration at the cellular level in entire lungs. This method reveals that TAM density was heterogeneous across tumours in the same animal, overall TAM density is different among separate pulmonary tumour models, nanotherapeutic drug delivery correlated with TAM heterogeneity, and successful response to CSF-1R blockade is characterized by enhanced TAM penetration throughout and within tumours.

Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.

The synthetic cryptocaryols A and B and a series of their analogues have been evaluated for their cytotoxicity and their ability to stabilize the tumor suppressor PDCD4. Cytotoxicities in the 3 to 30 μM range were found. Both the cytotoxicity and PDCD4 stabilizing ability were tolerant of large stereochemical changes to the molecule. Co-dosing studies with cryptocaryols A and B and several known cancer drugs showed no measuable enhancement in cancer drug cytotoxicity.

This chapter contains sections titled: Introduction De novo Approach to Carbohydrates An Iterative Pd-Catalyzed Glycosylation and Bidirectional Postglycosylation Application to the Synthesis of the Anthrax Tetrasaccharide CV of Michael F. Cuccarese CV of George A. O'Doherty References

Aminoglycosides, characterized by their toxic interaction with the bacterial ribosome, are also known for their effects on eukaryotic cells. While aminoglycosides are routinely used in the clinic as antibiotic agents, they are currently being evaluated therapeutically for their ability to rescue protein synthesis that is suppressed by nonsense‐mutation (e.g., in the case of cystic fibrosis). In this current study we aim to determine if the aminoglycoside read‐though effect on eukaryotic cells can be leveraged for use in cancer chemotherapy.Herein we describe our efforts to find the aminoglycoside read‐though effect on non‐small cell lung carcinoma cells (NCI‐H460) and it application to cancer cell chemotherapy. Specifically, these studies involve the search for sensitization effects of sub‐toxic doses of known read‐though inducing aminoglycosides in cancer cell cytotoxicity assays with known anticancer agents. For NCI‐H460 cancer cells, a dose dependent sensitization effect was found for known read‐though inducing aminoglycosides for some, but not all anticancer drugs. Studies aimed at the mode of action for sensitization due to aminoglycoside exposure is ongoing.

Over the years, considerable effort has been made toward the development of new synthetic routes to monosaccharides [1]. This interest came primarily from the medicinal chemistry community, as these new routes often provided access to unnatural sugars, which could be of use in structure–activity relationship (SAR) studies. In addition, the synthesis of monosaccharides, and in particular hexoses, has served as a challenge and a measuring stick to the synthetic organic community. Of particular interest are the routes to hexoses that start from achiral starting materials, where asymmetric catalysis is used to install the stereochemistry. In the synthetic organic community, these routes are described as "de novo" or "de novo asymmetric" routes to carbohydrates, whereas in the carbohydrate community, the term de novo takes up other meanings. For the purposes of this review, the term de novo asymmetric synthesis refers to the use of catalysis for the asymmetric synthesis of carbohydrates from achiral compounds [2]. This then precludes the inclusion of de novo process that produced sugars from molecules with preexisting chiral centers (e.g., Seeberger and Reißig) [3, 4].

A de novo asymmetric synthesis of alpha-ido-pyranosides, as well as several deoxy and amino variants, has been achieved. The procedure involves a palladium(0)-catalyzed glycosylation in combination with a Wharton rearrangement/epoxide-opening reaction sequence to access sugars with ido, manno, and colito stereochemistry as well as several azido analogues.

Aminoglycosides are broad-spectrum antibiotics that are used for the treatment of severe Gram-negative and Gram-positive bacterial infections. While bactericidal effects of aminoglycosides are due to binding to the 30S subunit of the bacterial ribosome, aminoglycosides can affect protein synthesis, intracellular calcium levels, and levels of reactive oxygen species (ROS) in eukaryotic cells. While aminoglycosides can be cytotoxic at high concentrations, our results show that at much lower doses, gentamicin can be implemented as a sensitizing agent for the NSCLC cell line NCI-H460, increasing the efficacy of camptothecin, digitoxin, and vinblastine in vitro. We have also established that this sensitization is reliant on the ROS response generated by gentamicin.