Quantitative MRI reveals heterogeneous impacts of treatment on diseased bone marrow in a mouse model of myelofibrosis.

PURPOSE: Analyzing bone marrow in the hematologic cancer myelofibrosis requires endpoint histology in mouse models and bone marrow biopsies in patients. These methods hinder the ability to monitor therapy over time. Preclinical studies typically begin treatment before mice develop myelofibrosis, unlike patients who begin therapy only after onset of disease. Using clinically relevant, quantitative MRI metrics allowed us to evaluate treatment in mice with established myelofibrosis. METHODS: We used chemical shift-encoded fat imaging, DWI, and magnetization transfer sequences to quantify bone marrow fat, cellularity, and macromolecular components in a mouse model of myelofibrosis. We monitored spleen volume, the established imaging marker for treatment, with anatomic MRI. After confirming bone marrow disease by MRI, we randomized mice to treatment with an approved drug (ruxolitinib or fedratinib) or an investigational agent, navitoclax, for 33 days. We measured the effects of therapy over time with bone marrow and spleen MRI. RESULTS: All treatments produced heterogeneous responses with improvements in bone marrow evident in subsets of individual mice in all treatment groups. Reductions in spleen volume commonly occurred without corresponding improvement in bone marrow. MRI revealed patterns associated with effective and ineffective responses to treatment in bone marrow and identified regional variations in efficacy within a bone. CONCLUSIONS: Quantitative MRI revealed modest, heterogeneous improvements in bone marrow disease when treating mice with established myelofibrosis. These results emphasize the value of bone marrow MRI to assess treatment in preclinical models and the potential to advance clinical trials for patients.

Both moderate- and high-intensity exercise training increase intramyocellular lipid droplet abundance and modify myocellular distribution in adults with obesity.

Exercise training modifies lipid metabolism in skeletal muscle, but the effect of exercise training on intramyocellular lipid droplet (LD) abundance, size, and intracellular distribution in adults with obesity remains elusive. This study compared high-intensity interval training (HIIT) with more conventional moderate-intensity continuous training (MICT) on intramyocellular lipid content, as well as LD characteristics (size and number) and abundance within the intramyofibrillar (IMF) and subsarcolemmal (SS) regions of type I and type II skeletal muscle fibers in adults with obesity. Thirty-six adults with obesity [body mass index (BMI) = 33 ± 3 kg/m2] completed 12 wk (4 days/wk) of either HIIT (10 × 1 min, 90% HRmax + 1-min active recovery; n = 19) or MICT (45-min steady-state exercise, 70% HRmax; n = 17), while on a weight-maintaining diet throughout training. Skeletal muscle biopsies were collected from the vastus lateralis before and after training, and intramyocellular lipid content and intracellular LD distribution were measured by immunofluorescence microscopy. Both MICT and HIIT increased total intramyocellular lipid content by more than 50% (P < 0.01), which was attributed to a greater LD number per µm2 in the IMF region of both type I and type II muscle fibers (P < 0.01). Our findings also suggest that LD lipophagy (autophagy-mediated LD degradation) may be transiently upregulated the day after the last exercise training session (P < 0.02 for both MICT and HIIT). In summary, exercise programs for adults with obesity involving either MICT or HIIT increased skeletal muscle LD abundance via a greater number of LDs in the IMF region of the myocyte, thereby providing more lipid in close proximity to the site of energy production during exercise.NEW & NOTEWORTHY In this study, 12 wk of either moderate-intensity continuous training (MICT) or high-intensity interval training (HIIT) enhanced skeletal muscle lipid abundance by increasing lipid droplet number within the intramyofibrillar (IMF) region of muscle. Because the IMF associates with high energy production during muscle contraction, this adaptation may enhance lipid oxidation during exercise. Despite differences in training intensity and energy expenditure between MICT and HIIT, their effects on muscle lipid abundance and metabolism were remarkably similar.

High-Throughput Cellular Heterogeneity Analysis in Cell Migration at the Single-Cell Level.

Cancer cell migration represents an essential step toward metastasis and cancer deaths. However, conventional drug discovery focuses on cytotoxic and growth-inhibiting compounds rather than inhibitors of migration. Drug screening assays generally measure the average response of many cells, masking distinct cell populations that drive metastasis and resist treatments. Here, this work presents a high-throughput microfluidic cell migration platform that coordinates robotic liquid handling and computer vision for rapidly quantifying individual cellular motility. Using this innovative technology, 172 compounds were tested and a surprisingly low correlation between migration and growth inhibition was found. Notably, many compounds were found to inhibit migration of most cells while leaving fast-moving subpopulations unaffected. This work further pinpoints synergistic drug combinations, including Bortezomib and Danirixin, to stop fast-moving cells. To explain the observed cell behaviors, single-cell morphological and molecular analysis were performed. These studies establish a novel technology to identify promising migration inhibitors for cancer treatment and relevant applications.

Computational modeling implicates protein scaffolding in p38 regulation of Akt.

Cells process environmental cues by activating intracellular signaling pathways with numerous interconnections and opportunities for cross-regulation. We employed a systems biology approach to investigate intersections of kinase p38, a context-dependent tumor suppressor or promoter, with Akt and ERK, two kinases known to promote cell survival, proliferation, and drug resistance in cancer. Using live, single cell microscopy, multiplexed fluorescent reporters of p38, Akt, and ERK activities, and a custom automated image-processing pipeline, we detected marked heterogeneity of signaling outputs in breast cancer cells stimulated with chemokine CXCL12 or epidermal growth factor (EGF). Basal activity of p38 correlated inversely with amplitude of Akt and ERK activation in response to either ligand. Remarkably, small molecule inhibitors of p38 immediately decreased basal activities of Akt and ERK but increased the proportion of cells with high amplitude ligand-induced activation of Akt signaling. To identify mechanisms underlying cross-talk of p38 with Akt signaling, we developed a computational model incorporating subcellular compartmentalization of signaling molecules by scaffold proteins. Dynamics of this model revealed that subcellular scaffolding of Akt accounted for observed regulation by p38. The model also predicted that differences in the amount of scaffold protein in a subcellular compartment captured the observed single cell heterogeneity in signaling. Finally, our model predicted that reduction in kinase signaling can be accomplished by both scaffolding and direct kinase inhibition. However, scaffolding inhibition can potentiate future kinase activity by redistribution of pathway components, potentially amplifying oncogenic signaling. These studies reveal how computational modeling can decipher mechanisms of cross-talk between the p38 and Akt signaling pathways and point to scaffold proteins as central regulators of signaling dynamics and amplitude.

Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer.

BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.

Single-cell RNA-sequencing of migratory breast cancer cells: discovering genes associated with cancer metastasis.

Considerable evidence suggests breast cancer metastasis arises from cells undergoing epithelial-to-mesenchymal-transition (EMT) and cancer stem-like cells (CSCs). Using a microfluidic device that enriches migratory breast cancer cells with enhanced capacity for tumor formation and metastasis, we identified genes differentially expressed in migratory cells by high-throughput single-cell RNA-sequencing. Migratory cells exhibited overall signatures of EMT and CSCs with variable expression of marker genes, and they retained expression profiles of EMT over time. With single-cell resolution, we discovered intermediate EMT states and distinct epithelial and mesenchymal sub-populations of migratory cells, indicating breast cancer cells can migrate rapidly while retaining an epithelial state. Migratory cells showed differential profiles for regulators of oxidative stress, mitochondrial morphology, and the proteasome, revealing potential vulnerabilities and unexpected consequences of drugs. We also identified novel genes correlated with cell migration and outcomes in breast cancer as potential prognostic biomarkers and therapeutic targets to block migratory cells in metastasis.

Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells.

Tumor-initiating cells, also designated as cancer stem cells, are proposed to constitute a subpopulation of malignant cells central to tumorigenesis, metastasis, and treatment resistance. We analyzed the activity of the proteasome, the primary organelle for targeted protein degradation, as a marker of tumor- and metastasis-initiating cells. Using human and mouse breast cancer cells expressing a validated fluorescent reporter, we found a small subpopulation of cells with low proteasome activity that divided asymmetrically to produce daughter cells with low or high proteasome activity. Breast cancer cells with low proteasome activity had greater local tumor formation and metastasis in immunocompromised and immunocompetent mice. To allow flexible labeling of cells, we also developed a new proteasome substrate based on HaloTag technology. Patient-derived glioblastoma cells with low proteasome activity measured by the HaloTag reporter show key phenotypes associated with tumor-initiating cells, including expression of a stem cell transcription factor, reconstitution of the original starting population, and enhanced neurosphere formation. We also show that patient-derived glioblastoma cells with low proteasome activity have higher frequency of tumor formation in mouse xenografts. These studies support proteasome function as a tool to investigate tumor- and metastasis-initiating cancer cells and a potential biomarker for outcomes in patients with several different cancers.

Host-derived CD8+ dendritic cells are required for induction of optimal graft-versus-tumor responses after experimental allogeneic bone marrow transplantation.

The graft-versus-tumor (GVT) effect after allogeneic hematopoietic cell transplantation (allo-HCT) represents an effective form of immunotherapy against many malignancies. Meaningful separation of the potentially curative GVT responses from graft-versus-host disease (GVHD), the most serious toxicity following T-cell replete allo-HCT, has been an elusive goal. GVHD is initiated by alloantigens, although both alloantigens and tumor-specific antigens (TSAs) initiate GVT responses. Emerging data have illuminated a role for antigen-presenting cells (APCs) in inducing alloantigen-specific responses. By using multiple clinically relevant murine models, we show that a specific subset of host-derived APCs-CD8(+) dendritic cells (DCs)-enhances TSA responses and is required for optimal induction of GVT. Stimulation of TLR3, which among host hematopoietic APC subsets is predominantly expressed on CD8(+) DCs, enhanced GVT without exacerbating GVHD. Thus, strategies that modulate host APC subsets without direct manipulation of donor T cells could augment GVT responses and enhance the efficacy of allo-HCT.

CXCR4-Targeted Nanocarriers for Triple Negative Breast Cancers.

CXCR4 is a cell membrane receptor that is overexpressed in triple-negative breast cancers and implicated in growth and metastasis of this disease. Using electrohydrodynamic cojetting, we prepared multicompartmental drug delivery carriers for CXCR4 targeting. The particles are comprised of a novel poly(lactide-co-glycolide) derivative that allows for straightforward immobilization of 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (Plerixafor), a small molecule with affinity for CXCR4. Targeted nanocarriers are selectively taken up by CXCR4-expressing cells and effectively block CXCR4 signaling. This study suggests that CXCR4 may be an effective target for nanocarrier-based therapies.

Characterizing heterogeneous single-cell dose responses computationally and experimentally using threshold inhibition surfaces and dose-titration assays.

Single cancer cells within a tumor exhibit variable levels of resistance to drugs, ultimately leading to treatment failures. While tumor heterogeneity is recognized as a major obstacle to cancer therapy, standard dose-response measurements for the potency of targeted kinase inhibitors aggregate populations of cells, obscuring intercellular variations in responses. In this work, we develop an analytical and experimental framework to quantify and model dose responses of individual cancer cells to drugs. We first explore the connection between population and single-cell dose responses using a computational model, revealing that multiple heterogeneous populations can yield nearly identical population dose responses. We demonstrate that a single-cell analysis method, which we term a threshold inhibition surface, can differentiate among these populations. To demonstrate the applicability of this method, we develop a dose-titration assay to measure dose responses in single cells. We apply this assay to breast cancer cells responding to phosphatidylinositol-3-kinase inhibition (PI3Ki), using clinically relevant PI3Kis on breast cancer cell lines expressing fluorescent biosensors for kinase activity. We demonstrate that MCF-7 breast cancer cells exhibit heterogeneous dose responses with some cells requiring over ten-fold higher concentrations than the population average to achieve inhibition. Our work reimagines dose-response relationships for cancer drugs in an emerging paradigm of single-cell tumor heterogeneity.

Growth signaling autonomy in circulating tumor cells aids metastatic seeding.

Self-sufficiency (autonomy) in growth signaling, the earliest recognized hallmark of cancer, is fueled by the tumor cell's ability to "secrete-and-sense" growth factors (GFs); this translates into cell survival and proliferation that is self-sustained by autocrine/paracrine secretion. A Golgi-localized circuitry comprised of two GTPase switches has recently been implicated in the orchestration of growth signaling autonomy. Using breast cancer cells that are either endowed or impaired (by gene editing) in their ability to assemble the circuitry for growth signaling autonomy, here we define the transcriptome, proteome, and phenome of such an autonomous state, and unravel its role during cancer progression. We show that autonomy is associated with enhanced molecular programs for stemness, proliferation, and epithelial-mesenchymal plasticity. Autonomy is both necessary and sufficient for anchorage-independent GF-restricted proliferation and resistance to anticancer drugs and is required for metastatic progression. Transcriptomic and proteomic studies show that autonomy is associated, with a surprising degree of specificity, with self-sustained epidermal growth factor receptor (EGFR)/ErbB signaling. Derivation of a gene expression signature for autonomy revealed that growth signaling autonomy is uniquely induced in circulating tumor cells (CTCs), the harshest phase in the life of tumor cells when it is deprived of biologically available epidermal growth factor (EGF). We also show that autonomy in CTCs tracks therapeutic response and prognosticates outcome. These data support a role for growth signaling autonomy in multiple processes essential for the blood-borne dissemination of human breast cancer.

NanoLuc reporter for dual luciferase imaging in living animals.

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate­independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc [NL]) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor ß signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as a new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development.

Near-infrared fluorescent proteins.

Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

Secreted CXCL12 (SDF-1) forms dimers under physiological conditions.

Chemokine CXCL12 (CXC chemokine ligand 12) signalling through CXCR (CXC chemokine receptor) 4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signalling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with GL (Gaussia luciferase) fusions to investigate dimerization of CXCL12 secreted from mammalian cells. Using column chromatography and GL complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signalling through Gαi and Akt, whereas dimeric CXCL12 more effectively promoted recruitment of ß-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumour xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiological conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signalling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.

Oscillatory ERK Signaling and Morphology Determine Heterogeneity of Breast Cancer Cell Chemotaxis via MEK-ERK and p38-MAPK Signaling Pathways.

Chemotaxis, regulated by oscillatory signals, drives critical processes in cancer metastasis. Crucial chemoattractant molecules in breast cancer, CXCL12 and EGF, drive the activation of ERK and Akt. Regulated by feedback and crosstalk mechanisms, oscillatory signals in ERK and Akt control resultant changes in cell morphology and chemotaxis. While commonly studied at the population scale, metastasis arises from small numbers of cells that successfully disseminate, underscoring the need to analyze processes that cancer cells use to connect oscillatory signaling to chemotaxis at single-cell resolution. Furthermore, little is known about how to successfully target fast-migrating cells to block metastasis. We investigated to what extent oscillatory networks in single cells associate with heterogeneous chemotactic responses and how targeted inhibitors block signaling processes in chemotaxis. We integrated live, single-cell imaging with time-dependent data processing to discover oscillatory signal processes defining heterogeneous chemotactic responses. We identified that short ERK and Akt waves, regulated by MEK-ERK and p38-MAPK signaling pathways, determine the heterogeneous random migration of cancer cells. By comparison, long ERK waves and the morphological changes regulated by MEK-ERK signaling, determine heterogeneous directed motion. This study indicates that treatments against chemotaxis in consider must interrupt oscillatory signaling.

Microfluidic single-cell migration chip reveals insights into the impact of extracellular matrices on cell movement.

Cell migration is a complex process that plays a crucial role in normal physiology and pathologies such as cancer, autoimmune diseases, and mental disorders. Conventional cell migration assays face limitations in tracking a large number of individual migrating cells. To address this challenge, we have developed a high-throughput microfluidic cell migration chip, which seamlessly integrates robotic liquid handling and computer vision to swiftly monitor the movement of 3200 individual cells, providing unparalleled single-cell resolution for discerning distinct behaviors of the fast-moving cell population. This study focuses on the ECM's role in regulating cellular migration, utilizing this cutting-edge microfluidic technology to investigate the impact of ten different ECMs on triple-negative breast cancer cell lines. We found that collagen IV, collagen III, and collagen I coatings were the top enhancers of cell movement. Combining these ECMs increased cell motility, but the effect was sub-additive. Furthermore, we examined 87 compounds and found that while some compounds inhibited migration on all substrates, significantly distinct effects on differently coated substrates were observed, underscoring the importance of considering ECM coating. We also utilized cells expressing a fluorescent actin reporter and observed distinct actin structures in ECM-interacting cells. ScRNA-Seq analysis revealed that ECM coatings induced EMT and enhanced cell migration. Finally, we identified genes that were particularly up-regulated by collagen IV and the selective inhibitors successfully blocked cell migration on collagen IV. Overall, the study provides insights into the impact of various ECMs on cell migration and dynamics of cell movement with implications for developing therapeutic strategies to combat diseases related to cell motility.

Regulation of DNA damage response by trimeric G-proteins.

Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of the two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DSBs hinges on GIV/Girdin, a guanine nucleotide-exchange modulator of heterotrimeric Giα•ßγ protein. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1's BRCT-modules via both phospho-dependent and -independent mechanisms. Using another non-overlapping C-terminal motif GIV binds and activates Gi and enhances the "free" Gßγ→PI-3-kinase→Akt pathway, which promotes survival and is known to suppress HR, favor NHEJ. Absence of GIV, or loss of either of its C-terminal motifs enhanced cell death upon genotoxic stress. Because GIV selectively binds other BRCT-containing proteins suggests that G-proteins may fine-tune sensing, repair, and survival after diverse types of DNA damage.

A Multiomic Analysis Reveals How Breast Cancers Disseminated to the Bone Marrow Acquire Aggressive Phenotypes through Tumor-Stroma Tunnels.

Estrogen receptor-positive (ER+) breast cancer commonly disseminates to bone marrow, where interactions with mesenchymal stromal cells (MSCs) shape disease trajectory. We modeled these interactions with tumor-MSC co-cultures and used an integrated transcriptome-proteome-network- analyses workflow to identify a comprehensive catalog of contact-induced changes. Induced genes and proteins in cancer cells, some borrowed and others tumor-intrinsic, were not recapitulated merely by conditioned media from MSCs. Protein-protein interaction networks revealed the rich connectome between 'borrowed' and 'intrinsic' components. Bioinformatic approaches prioritized one of the 'borrowed' components, CCDC88A /GIV, a multi-modular metastasis-related protein which has recently been implicated in driving one of the hallmarks of cancers, i.e., growth signaling autonomy. MSCs transferred GIV protein to ER+ breast cancer cells (that lack GIV) through tunnelling nanotubes via connexin (Cx)43-facilitated intercellular transport. Reinstating GIV alone in GIV-negative breast cancer cells reproduced ∼20% of both the 'borrowed' and the 'intrinsic' gene induction patterns from contact co-cultures; conferred resistance to anti-estrogen drugs; and enhanced tumor dissemination. Findings provide a multiomic insight into MSC→tumor cell intercellular transport and validate how transport of one such candidate, GIV, from the haves (MSCs) to have-nots (ER+ breast cancer) orchestrates aggressive disease states.

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system.

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust ßarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced ßarr-coupling compared to CXCR7. These two receptors induce distinct ßarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for ßarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.

The CXCL12/CXCR4/ACKR3 Signaling Axis Regulates PKM2 and Glycolysis.

In response to CXCL12, CXCR4 and ACKR3 both recruit ß-arrestin 2, regulating the assembly of interacting proteins that drive signaling and contribute to the functions of both receptors in cancer and multiple other diseases. A prior proteomics study revealed that ß-arrestin 2 scaffolds pyruvate kinase M2 (PKM2), an enzyme implicated in shifting cells to glycolytic metabolism and poor prognosis in cancer. We hypothesized that CXCL12 signaling regulates PKM2 protein interactions, oligomerization, and glucose metabolism. We used luciferase complementation in cell-based assays and a tumor xenograft model of breast cancer in NSG mice to quantify how CXCR4 and ACKR3 change protein interactions in the ß-arrestin-ERK-PKM2 pathway. We also used mass spectrometry to analyze the effects of CXCL12 on glucose metabolism. CXCL12 signaling through CXCR4 and ACKR3 stimulated protein interactions among ß-arrestin 2, PKM2, ERK2, and each receptor, leading to the dissociation of PKM2 from ß-arrestin 2. The activation of both receptors reduced the oligomerization of PKM2, reflecting a shift from tetramers to dimers or monomers with low enzymatic activity. Mass spectrometry with isotopically labeled glucose showed that CXCL12 signaling increased intermediate metabolites in glycolysis and the pentose phosphate pathway, with ACKR3 mediating greater effects. These data establish how CXCL12 signaling regulates PKM2 and reprograms cellular metabolism.