Targeting CCL2/CCR2 Signaling Overcomes MEK Inhibitor Resistance in Acute Myeloid Leukemia.

PURPOSE: Emerging evidence underscores the critical role of extrinsic factors within the microenvironment in protecting leukemia cells from therapeutic interventions, driving disease progression, and promoting drug resistance in acute myeloid leukemia (AML). This finding emphasizes the need for the identification of targeted therapies that inhibit intrinsic and extrinsic signaling to overcome drug resistance in AML. EXPERIMENTAL DESIGN: We performed a comprehensive analysis utilizing a cohort of ∼300 AML patient samples. This analysis encompassed the evaluation of secreted cytokines/growth factors, gene expression, and ex vivo drug sensitivity to small molecules. Our investigation pinpointed a notable association between elevated levels of CCL2 and diminished sensitivity to the MEK inhibitors (MEKi). We validated this association through loss-of-function and pharmacologic inhibition studies. Further, we deployed global phosphoproteomics and CRISPR/Cas9 screening to identify the mechanism of CCR2-mediated MEKi resistance in AML. RESULTS: Our multifaceted analysis unveiled that CCL2 activates multiple prosurvival pathways, including MAPK and cell-cycle regulation in MEKi-resistant cells. Employing combination strategies to simultaneously target these pathways heightened growth inhibition in AML cells. Both genetic and pharmacologic inhibition of CCR2 sensitized AML cells to trametinib, suppressing proliferation while enhancing apoptosis. These findings underscore a new role for CCL2 in MEKi resistance, offering combination therapies as an avenue to circumvent this resistance. CONCLUSIONS: Our study demonstrates a compelling rationale for translating CCL2/CCR2 axis inhibitors in combination with MEK pathway-targeting therapies, as a potent strategy for combating drug resistance in AML. This approach has the potential to enhance the efficacy of treatments to improve AML patient outcomes.

Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 µg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

Imidazolone as an Amide Bioisostere in the Development of ß-1,3-N-Acetylglucosaminyltransferase 2 (B3GNT2) Inhibitors.

B3GNT2 is responsible for elongation of cell surface long-chain polylactosamine, which influences the regulation of the immune response, making it an attractive target for immunomodulation. In the development of amide containing B3GNT2 inhibitors guided by structure-based drug design, imidazolones were found to successfully serve as amide bioisosteres. This novel imidazolone isosteric strategy alleviated torsional strain of the amide bond on binding to B3GNT2 and improved potency, isoform selectivity, as well as certain physicochemical and pharmacokinetic properties. Herein, we present the synthesis, SAR, X-ray cocrystal structures, and in vivo PK properties of imidazol-4-ones in the context of B3GNT2 inhibition.

Proteomic and phosphoproteomic measurements enhance ability to predict ex vivo drug response in AML.

Acute Myeloid Leukemia (AML) affects 20,000 patients in the US annually with a five-year survival rate of approximately 25%. One reason for the low survival rate is the high prevalence of clonal evolution that gives rise to heterogeneous sub-populations of leukemic cells with diverse mutation spectra, which eventually leads to disease relapse. This genetic heterogeneity drives the activation of complex signaling pathways that is reflected at the protein level. This diversity makes it difficult to treat AML with targeted therapy, requiring custom patient treatment protocols tailored to each individual's leukemia. Toward this end, the Beat AML research program prospectively collected genomic and transcriptomic data from over 1000 AML patients and carried out ex vivo drug sensitivity assays to identify genomic signatures that could predict patient-specific drug responses. However, there are inherent weaknesses in using only genetic and transcriptomic measurements as surrogates of drug response, particularly the absence of direct information about phosphorylation-mediated signal transduction. As a member of the Clinical Proteomic Tumor Analysis Consortium, we have extended the molecular characterization of this cohort by collecting proteomic and phosphoproteomic measurements from a subset of these patient samples (38 in total) to evaluate the hypothesis that proteomic signatures can improve the ability to predict response to 26 drugs in AML ex vivo samples. In this work we describe our systematic, multi-omic approach to evaluate proteomic signatures of drug response and compare protein levels to other markers of drug response such as mutational patterns. We explore the nuances of this approach using two drugs that target key pathways activated in AML: quizartinib (FLT3) and trametinib (Ras/MEK), and show how patient-derived signatures can be interpreted biologically and validated in cell lines. In conclusion, this pilot study demonstrates strong promise for proteomics-based patient stratification to assess drug sensitivity in AML.

A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation.

The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-ß) from latent complexes via integrin-αV activation. Using acute injury models in two different tissues, we found that by inducing TGF-ß activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.

Isolation and Culture of Murine Hepatic Stellate Cells.

Hepatic stellate cells (HSCs), alternatively known as liver pericytes, can differentiate into myofibroblasts and secrete extra-cellular matrix components, thereby promoting wound healing and fibrosis. Studying HSCs can provide insights into the pathological mechanisms governing these processes. HSC isolation methods typically comprise of enzymatic digestion followed by density gradient centrifugation and/or Fluorescent Activated Cell Sorting (FACS) mediated sorting. In this protocol, we describe a step-wise method for HSC isolation that utilizes Pronase and Collagenase for enzymatic tissue dissociation, followed by an Optiprep based density gradient centrifugation. The isolation can be performed using common media and buffers, and without the use of any special equipment for liver perfusion and HSC isolation. The technique yields ex vivo HSCs, suitable for use in assays.