AMPK inhibition sensitizes acute leukemia cells to BH3 mimetic-induced cell death.

BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.

PARP Inhibitors and Myeloid Neoplasms: A Double-Edged Sword.

Despite recent discoveries and therapeutic advances in aggressive myeloid neoplasms, there remains a pressing need for improved therapies. For instance, in acute myeloid leukemia (AML), while most patients achieve a complete remission with conventional chemotherapy or the combination of a hypomethylating agent and venetoclax, de novo or acquired drug resistance often presents an insurmountable challenge, especially in older patients. Poly(ADP-ribose) polymerase (PARP) enzymes, PARP1 and PARP2, are involved in detecting DNA damage and repairing it through multiple pathways, including base excision repair, single-strand break repair, and double-strand break repair. In the context of AML, PARP inhibitors (PARPi) could potentially exploit the frequently dysfunctional DNA repair pathways that, similar to deficiencies in homologous recombination in BRCA-mutant disease, set the stage for cell killing. PARPi appear to be especially effective in AML with certain gene rearrangements and molecular characteristics (RUNX1-RUNX1T1 and PML-RARA fusions, FLT3- and IDH1-mutated). In addition, PARPi can enhance the efficacy of other agents, particularly alkylating agents, TOP1 poisons, and hypomethylating agents, that induce lesions ordinarily repaired via PARP1-dependent mechanisms. Conversely, emerging reports suggest that long-term treatment with PARPi for solid tumors is associated with an increased incidence of myelodysplastic syndrome (MDS) and AML. Here, we (i) review the pre-clinical and clinical data on the role of PARPi, specifically olaparib, talazoparib, and veliparib, in aggressive myeloid neoplasms and (ii) discuss the reported risk of MDS/AML with PARPi, especially as the indications for PARPi use expand to include patients with potentially curable cancer.

Engaging the Innate and Adaptive Antitumor Immune Response in Lymphoma.

Immunotherapy has emerged as a powerful therapeutic strategy for many malignancies, including lymphoma. As in solid tumors, early clinical trials have revealed that immunotherapy is not equally efficacious across all lymphoma subtypes. For example, immune checkpoint inhibition has a higher overall response rate and leads to more durable outcomes in Hodgkin lymphomas compared to non-Hodgkin lymphomas. These observations, combined with a growing understanding of tumor biology, have implicated the tumor microenvironment as a major determinant of treatment response and prognosis. Interactions between lymphoma cells and their microenvironment facilitate several mechanisms that impair the antitumor immune response, including loss of major histocompatibility complexes, expression of immunosuppressive ligands, secretion of immunosuppressive cytokines, and the recruitment, expansion, and skewing of suppressive cell populations. Accordingly, treatments to overcome these barriers are being rapidly developed and translated into clinical trials. This review will discuss the mechanisms of immune evasion, current avenues for optimizing the antitumor immune response, clinical successes and failures of lymphoma immunotherapy, and outstanding hurdles that remain to be addressed.

Engineering reversible cell-cell interactions with chemical biology.

Techniques to direct cell-cell interactions have advanced our understanding of fundamental biology and opened new avenues in tissue engineering, regenerative medicine, and immunotherapy. This is often achieved by introducing new targeting ligands to the cell membrane, which can be accomplished through both genetic and nongenetic approaches. While both offer advantages, nongenetic modifications tend to be faster to produce, innocuous to the modified cell, and potentially reversible. This chapter will outline nongenetic methods that have been used to control intercellular interactions-namely hydrophobic insertion, chemical modification, liposome fusion, metabolic engineering, and enzymatic remodeling-and provide protocols that can serve as a starting point for future applications.

Engineering reversible cell-cell interactions using enzymatically lipidated chemically self-assembled nanorings.

Multicellular biology is dependent on the control of cell-cell interactions. These concepts have begun to be exploited for engineering of cell-based therapies. Herein, we detail the use of a multivalent lipidated scaffold for the rapid and reversible manipulation of cell-cell interactions. Chemically self-assembled nanorings (CSANs) are formed via the oligomerization of bivalent dihydrofolate reductase (DHFR2) fusion proteins using a chemical dimerizer, bis-methotrexate. With targeting proteins fused onto the DHFR2 monomers, the CSANs can target specific cellular antigens. Here, anti-EGFR or anti-EpCAM fibronectin-DHFR2 monomers incorporating a CAAX-box sequence were enzymatically prenylated, then assembled into the corresponding CSANs. Both farnesylated and geranylgeranylated CSANs efficiently modified the cell surface of lymphocytes and remained bound to the cell surface with a half-life of >3 days. Co-localization studies revealed a preference for the prenylated nanorings to associate with lipid rafts. The presence of antigen targeting elements in these bifunctional constructs enabled them to specifically interact with target cells while treatment with trimethoprim resulted in rapid CSAN disassembly and termination of the cell-cell interactions. Hence, we were able to determine that activated PBMCs modified with the prenylated CSANs caused irreversible selective cytotoxicity toward EGFR-expressing cells within 2 hours without direct engagement of CD3. The ability to disassemble these nanostructures in a temporally controlled manner provides a unique platform for studying cell-cell interactions and T cell-mediated cytotoxicity. Overall, antigen-targeted prenylated CSANs provide a general approach for the regulation of specific cell-cell interactions and will be valuable for a plethora of fundamental and therapeutic applications.

Multivalent Ligand Binding to Cell Membrane Antigens: Defining the Interplay of Affinity, Valency, and Expression Density.

Nature uses multivalency to govern many biological processes. The development of macromolecular and cellular therapies has largely been dependent on engineering similar polyvalent interactions to enable effective targeting. Such therapeutics typically utilize high-affinity binding domains that have the propensity to recognize both antigen-overexpressing tumors and normal-expressing tissues, leading to "on-target, off-tumor" toxicities. One strategy to improve these agents' selectivity is to reduce the binding affinity, such that biologically relevant interactions between the therapeutic and target cell will only exist under conditions of high avidity. Preclinical studies have validated this principle of avidity optimization in the context of chimeric antigen receptor (CAR) T cells; however, a rigorous analysis of this approach in the context of soluble multivalent targeting scaffolds has yet to be undertaken. Using a modular protein nanoring capable of displaying ≤8 fibronectin domains with engineered specificity for a model antigen, epithelial cell adhesion molecule (EpCAM), this study demonstrates that binding affinity and ligand valency can be optimized to afford discrimination between EpCAMHigh (2.8-3.8 × 106 antigens/cell) and EpCAMLow (5.2 × 104 to 2.2 × 105 antigens/cell) tissues both in vitro and in vivo.

Programming Cell-Cell Interactions through Non-genetic Membrane Engineering.

The ability to direct targeted intercellular interactions has the potential to enable and expand the use of cell-based therapies for regenerative medicine, tissue engineering, and immunotherapy. While genetic engineering approaches have proven effective, these techniques are not amenable to all cell types and often yield permanent modifications with potentially long-lasting adverse effects, restricting their application. To circumvent these limitations, there is intense interest in developing non-genetic methods to modify cell membranes with functional groups that will enable the recognition of target cells. While many such techniques have been developed, relatively few have been applied to directing specific cell-cell interactions. This review details these non-genetic membrane engineering approaches-namely, hydrophobic membrane insertion, chemical modification, liposome fusion, metabolic engineering, and enzymatic remodeling-and summarizes their major applications. Based on this analysis, perspective is provided on the ideal features of these systems with an emphasis on the potential for clinical translation.

Eradication of Established Tumors by Chemically Self-Assembled Nanoring Labeled T Cells.

Our laboratory has developed chemically self-assembled nanorings (CSANs) as prosthetic antigen receptors (PARs) for the nongenetic modification of T cell surfaces. PARs have been successfully employed in vitro to activate T cells for the selective killing of leukemia cells. However, PAR efficacy has yet to be evaluated in vivo or against solid tumors. Therefore, we developed bispecific PARs that selectively target the human CD3 receptor and human epithelial cell adhesion molecule (EpCAM), which is overexpressed on multiple carcinomas and cancer stem cells. The αEpCAM/αCD3 PARs were found to stably bind T cells for >4 days, and treating EpCAM+ MCF-7 breast cancer cells with αEpCAM/αCD3 PAR-functionalized T cells resulted in the induction of IL-2, IFN-γ, and MCF-7 cytotoxicity. Furthermore, an orthotopic breast cancer model validated the ability of αEpCAM/αCD3 PAR therapy to direct T cell lytic activity toward EpCAM+ breast cancer cells in vivo, leading to tumor eradication. In vivo biodistribution studies demonstrated that PAR-T cells were formed in vivo and persist for over 48 h with rapid accumulation in tumor tissue. Following PAR treatment, the production of IL-2, IFN-γ, IL-6, and TNF-α could be significantly reduced by an infusion of clinically relevant concentrations of the FDA-approved antibiotic, trimethoprim, signaling pharmacologic PAR deactivation. Importantly, CSANs did not induce naïve T cell activation and thus exhibit a limited potential to induce naïve T cell anergy. In addition, murine immunogenicity studies demonstrated that CSANs do not induce a significant antibody response nor do they activate splenic cells. Collectively, our results demonstrate that bispecific CSANs are able to nongenetically generate reversibly modified T cells that are capable of eradicating targeted solid tumors.

Tunable Supramolecular Assemblies from Amphiphilic Nucleoside Phosphoramidate Nanofibers by Enzyme Activation.

Enzymes possess unique qualities that make them ideal regulators of supramolecular assembly. They are uniquely sensitive to biomolecules and biological compartments, catalytic in effecting chemical reactions, and present a biocompatible and degradable platform for assembly regulation. We demonstrate the novel utility of Histidine Triad Nucleotide Binding Protein 1 (HINT1) in regulating supramolecular hydrogel formation. We synthesized nucleoside-phosphoramidate-functionalized self-assembling peptides that we observed to form nanofibers. We found HINT1's catalytic hydrolysis of the nucleoside phosphoramidate moieties within the nanofiber structures to induce nanofiber organization and higher ordered assembly. The role of HINT1 in effecting this structural change was confirmed with experiments utilizing a high-affinity HINT1 inhibitor and catalytically dead HINT1 mutant. In addition, the kinetics and morphology of hydrogel formation were found to be dependent on the structure of the released nucleoside monophosphate. This work highlights the self-assembly of phosphoramidate nanofibers and their higher organization triggered by HINT1 enzymatic activity.

Engineering Reversible Cell-Cell Interactions with Lipid Anchored Prosthetic Receptors.

Membrane-engineered cells displaying antigen-targeting ligands are useful as both scientific tools and clinical therapeutics. While genetically encoded artificial receptors have proven efficacious, their scope remains limited, as this approach is not amenable to all cell types and the modification is often permanent. Our group has developed a nongenetic method to rapidly, stably, and reversibly modify any cell membrane with a chemically self-assembled nanoring (CSAN) that can function as a prosthetic receptor. Bifunctional CSANs displaying epithelial cell adhesion molecule (EpCAM)-targeted fibronectin domains were installed on the cell membrane through hydrophobic insertion and remained stably bound for ≥72 h in vitro. These CSAN-labeled cells were capable of recognizing EpCAM-expressing target cells, forming intercellular interactions that were subsequently reversed by disassembling the nanoring with the FDA-approved antibiotic, trimethoprim. This study demonstrates the use of this system to engineer cell surfaces with prosthetic receptors capable of directing specific and reversible cell-cell interactions.

Titratable Avidity Reduction Enhances Affinity Discrimination in Mammalian Cellular Selections of Yeast-Displayed Ligands.

Yeast surface display selections against mammalian cell monolayers have proven effective in isolating proteins with novel binding activity. Recent advances in this technique allow for the recovery of clones with even micromolar binding affinities. However, no efficient method has been shown for affinity-based selection in this context. This study demonstrates the effectiveness of titratable avidity reduction using dithiothreitol to achieve this goal. A series of epidermal growth factor receptor binding fibronectin domains with a range of affinities are used to quantitatively identify the number of ligands per yeast cell that yield the strongest selectivity between strong, moderate, and weak affinities. Notably, reduction of ligand display to 3,000-6,000 ligands per yeast cell of a 2 nM binder yields 16-fold better selectivity than that to a 17 nM binder. These lessons are applied to affinity maturation of an EpCAM-binding fibronectin population, yielding an enriched pool of ligands with significantly stronger affinity than that of an analogous pool sorted by standard cellular selection methods. Collectively, this study offers a facile approach for affinity selection of yeast-displayed ligands against full-length cellular targets and demonstrates the effectiveness of this method by generating EpCAM-binding ligands that are promising for further applications.